Johanne Seguin

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.

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.

Nanoemulsion formulation of fisetin improves bioavailability and antitumour activity in mice.

The natural flavonoid fisetin (3,3,4,7-tetrahydroxyflavone) has shown antitumour activity but its administration is complicated by its low water solubility. Our aim was to incorporate fisetin into a nanoemulsion to improve its pharmacokinetics and therapeutic efficacy. Solubility and emulsification tests allowed to develop an optimal nanoemulsion composed of Miglyol 812N/Labrasol/Tween 80/Lipoid E80/water (10%/10%/2.5%/1.2%/76.3%). The nanoemulsion had an oil droplet diameter of 153 ± 2 nm, a negative zeta potential (-28.4 ± 0.6 mV) and a polydispersity index of 0.129. The nanoemulsion was stable at 4 °C for 30 days, but phase separation occurred at 20 °C. Pharmacokinetic studies in mice revealed that the fisetin nanoemulsion injected intravenously (13 mg/kg) showed no significant difference in systemic exposure compared to free fisetin. However, when the fisetin nanoemulsion was administered intraperitoneally, a 24-fold increase in fisetin relative bioavailability was noted, compared to free fisetin. Additionally, the antitumour activity of the fisetin nanoemulsion in Lewis lung carcinoma bearing mice occurred at lower doses (36.6 mg/kg) compared to free fisetin (223 mg/kg). In conclusion, we have developed a stable nanoemulsion of fisetin and have shown that it could improve its relative bioavailability and antitumour activity.

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.

Improved antiangiogenic and antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide in Lewis lung carcinoma-bearing mice.

PurposeThe natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study, we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo.MethodsFisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC-bearing mice treated with fisetin and/or cyclophosphamide (CPA).ResultsThe fisetin IC50 was 59xa0μM for LLC and 77xa0μM for EC cells, compared to 210xa0μM for normal NIH 3T3 cells (24xa0h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22–44xa0μM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC-bearing mice, fisetin produced a 67% tumour growth inhibition (223xa0mg/kg, intraperitoneal), similar to the 66% produced by low-dose CPA (30xa0mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination.ConclusionsWe have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but also can markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours.

Bioavailability of Polyphenol Liposomes: A Challenge Ahead

Dietary polyphenols, including flavonoids, have long been recognized as a source of important molecules involved in the prevention of several diseases, including cancer. However, because of their poor bioavailability, polyphenols remain difficult to be employed clinically. Over the past few years, a renewed interest has been devoted to the use of liposomes as carriers aimed at increasing the bioavailability and, hence, the therapeutic benefits of polyphenols. In this paper, we review the causes of the poor bioavailability of polyphenols and concentrate on their liposomal formulations, which offer a means of improving their pharmacokinetics and pharmacodynamics. The problems linked to their development and their potential therapeutic advantages are reviewed. Future directions for liposomal polyphenol development are suggested.

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.

Development of a liposomal formulation of the natural flavonoid fisetin.

The natural flavonoid fisetin (3,3,4,7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration, taking into account the availability and cost of the various components. Several methods were evaluated such as probe sonication, homogeneization, film hydration and lipid cake formation. A selection of lipid and lipid-PEG was also performed via their incorporation in different formulations based on the size of the liposomes, their polydispersity index (PDI) and the fisetin encapsulation yield. An optimal liposomal formulation was developed with P90G and DODA-GLY-PEG2000, possessing a diameter in the nanometer scale (175nm), a high homogeneity (PDI 0.12) and a high fisetin encapsulation (73%). Fisetin liposomes were stable over 59 days for their particle diameter and still retained 80% of their original fisetin content on day 32. Moreover, liposomal fisetin retained the cytotoxicity and typical morphological effect of free fisetin in different tumour and endothelial cell lines. In conclusion, based on its physico-chemical properties and retention of fisetin biological effects, the developed liposomal fisetin preparation is therefore suitable for in vivo administration.

Chemically engineered persistent luminescence nanoprobes for bioimaging

Imaging nanoprobes are a group of nanosized agents developed for providing improved contrast for bioimaging. Among various imaging probes, optical sensors capable of following biological events or progresses at the cellular and molecular levels are actually actively developed for early detection, accurate diagnosis, and monitoring of the treatment of diseases. The optical activities of nanoprobes can be tuned on demand by chemists by engineering their composition, size and surface nature. This review will focus on researches devoted to the conception of nanoprobes with particular optical properties, called persistent luminescence, and their use as new powerful bioimaging agents in preclinical assays.

Synthesis and functionalization of persistent luminescence nanoparticles with small molecules and evaluation of their targeting ability

We have recently reported the design and use of inorganic nanoparticles with persistent luminescence properties. Such nanoparticles can be excited with a UV lamp for 2min and emit light in the near-infrared area for dozen of minutes without any further excitation. This property is of particular interest for small animal optical imaging, since it avoids the autofluorescence of endogenous fluorophores which is one major problem encountered when using fluorescent probes. We report herein the synthesis of persistent luminescence nanoparticles (PLNPs) and their functionalization with two small targeting molecules: biotin and Rak-2. We provide characterization of each PLNP as well as preliminary evidence of the ability of PLNP-PEG-Biotin to target streptavidin and PLNP-PEG-Rak-2 to bind prostate cancer cells in vitro.