Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model.

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy.

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at -80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Mucoadhesive thermosensitive hydrogel for the intra-tumoral delivery of immunomodulatory agents, in vivo evidence of adhesion by means of non-invasive imaging techniques.

Intratumoral injection of biocompatible gels is increasingly used for the sustained delivery of drugs and vaccines to enhance the anti-cancer immune response. Granulocyte-macrophage colony stimulating factor (GM-CSF) has become an attractive adjuvant thanks to its ability to boost the antitumor immune response by inducing proliferation, maturation and migration of the dendritic-cells (DCs) and the differentiation of lymphocytes. Killed Mycobacteria, such as Heat-killed Mycobacterium tuberculosis (HKMT) have been used in several studies as TLR-2 agonist to increase maturation of DCs. In this study, we designed a mucoadhesive thermosensitive formulation for the local delivery of GM-CSF and HKMT in order to enhance DCs activation and improve the local antitumor immune response. This formulation was selected based on its elastic and mucoadhesive properties obtained thanks to rheological studies. More importantly, intratumoral residence time of the labelled gel and protein were evidenced by means of MRI and non invasive in vivo optical imaging. Then, the efficacy of the combination of immunomodulators loaded thermogel was demonstated in vitro and in vivo. The selected thermogel exhibits rheological properties which confer a good elasticity and increased residence time of the immunostimulatory agents in the tumor, thus increasing the recruitment of DCs and T cytotoxic CD8+ lymphocytes.

Gadolinium and Platinum in Tandem: Real-time Multi-Modal Monitoring of Drug Delivery by MRI and Fluorescence Imaging.

A novel dual-imaging cisplatin-carrying molecular cargo capable of performing simultaneous optical and MR imaging is reported herein. This long-lasting MRI contrast agent (r1 relaxivity of 23.4 mM-1s-1 at 3T, 25 oC) is a photo-activated cisplatin prodrug (PtGdL) which enables real-time monitoring of anti-cancer efficacy. PtGdL is a model for monitoring the drug delivery and anti-cancer efficacy by MRI with a much longer retention time (24 hours) in several organs such as renal cortex and spleen than GdDOTA and its motif control GdL. Upon complete release of cisplatin, all PtGdL is converted to GdL enabling subsequent MRI analyses of therapy efficacy within its reasonably short clearance time of 4 hours. There is also responsive fluorescence enhancement for monitoring by photon-excitation.

Long-term toxicological effects of persistent luminescence nanoparticles after intravenous injection in mice.

The ZnGa1.995Cr0.005O4 persistent luminescence nanoparticles offer the promise of revolutionary tools for biological imaging with applications such as cell tracking or tumor detection. They can be re-excited through living tissues by visible photons, allowing observations without any time constraints and avoiding the undesirable auto-fluorescence signals observed when fluorescent probes are used. Despite all these advantages, their uses demand extensive toxicological evaluation and control. With this purpose, mice were injected with a single intravenous administration of hydroxylated or PEGylated persistent luminescence nanoparticles at different concentrations and then a set of standard tests were carried out 1day, 1 month and 6 months after the administration. High concentrations of hydroxylated nanoparticles generate structural alterations at histology level, endoplasmic reticulum damage and oxidative stress in liver, as well as rising in white blood cells counts. A mechanism involving the endoplasmic reticulum damage could be the responsible of the observed injuries in case of ZGO-OH. On the contrary, no toxicological effects related to PEGylated nanoprobes treatment were noted during our in vivo experiments, denoting the protective effect of PEG-functionalization and thereby, their potential as biocompatible in vivo diagnostic probes.

Photo-stimulation of persistent luminescence nanoparticles enhances cancer cells death.

Persistent luminescence nanoparticles made of ZnGa1.995Cr0.005O4 (ZGO-NPs) are innovative nanomaterials that emit photons during long periods of time after the end of the excitation, allowing their use as diagnosis probes for in vivo optical imaging. During the excitation process, a part of the energy is stored in traps to further emit photons over long time. However, we observed in this study that some of the energy reduces molecular oxygen to produce reactive oxygen species (ROS). Following this observation, theoxidative stress induction and cytotoxic effects of these NPs were investigated on human breast cancer cells. The results indicate that ROS production was stimulated by exposition of the hydroxylated ZGO-NPs to UV or visible light, and the oxidative stress induced in cells after internalization can be directly correlated to their dose-dependent inhibition of cell viability. On the contrary, PEGylated ZGONPs were not uptaken by cells and have no effect on the production of ROS. Thus, the cell viability was not altered by these nanoparticles. This study reveals the importance of considering light irradiation and surface coating of luminescent nanoparticles toxicity which open new perspectives for their use in photodynamic therapy.

Mesoporous persistent nanophosphors for in vivo optical bioimaging and drug-delivery.

Based upon the ambitious idea that one single particle could serve multiple purposes at the same time, the combination and simultaneous use of imaging and therapeutics has lately arisen as one of the most promising prospects among nanotechnologies directed toward biomedical applications. Intended for both therapeutics and diagnostics in vivo, highly complex nanostructures were specifically designed to simultaneously act as optical imaging probes and delivery vehicles. Yet, such multifunctional photonic nanoplatforms usually exploit fluorescence phenomena which require constant excitation light through biological tissues and thus significantly reduce the detection sensitivity due to the autofluorescence from living animals. In order to overcome this critical issue, the present article introduces a novel multifunctional agent based on persistent luminescence mesoporous nanoparticles. Being composed of a hybrid chromium-doped zinc gallate core/mesoporous silica shell architecture, we show that this nanotechnology can be used as an efficient doxorubicin-delivery vehicle presenting a higher cytotoxicity toward U87MG cells than its unloaded counterpart in vitro. In addition, we demonstrate that a persistent luminescence signal from these doxorubicin-loaded mesoporous nanophosphors opens a new way to highly sensitive detection in vivo, giving access to the real-time biodistribution of the carrier without any autofluorescence from the animal tissues. This new persistent luminescence-based hybrid nanotechnology can be easily applied to the delivery of any therapeutic agent, thus constituting a versatile and sensitive optical nanotool dedicated to both therapeutic and diagnostic applications in vivo.

The in vivo activation of persistent nanophosphors for optical imaging of vascularization, tumours and grafted cells.

Optical imaging for biological applications requires more sensitive tools. Near-infrared persistent luminescence nanoparticles enable highly sensitive in vivo optical detection and complete avoidance of tissue autofluorescence. However, the actual generation of persistent luminescence nanoparticles necessitates ex vivo activation before systemic administration, which prevents long-term imaging in living animals. Here, we introduce a new generation of optical nanoprobes, based on chromium-doped zinc gallate, whose persistent luminescence can be activated in vivo through living tissues using highly penetrating low-energy red photons. Surface functionalization of this photonic probe can be adjusted to favour multiple biomedical applications such as tumour targeting. Notably, we show that cells can endocytose these nanoparticles in vitro and that, after intravenous injection, we can track labelled cells in vivo and follow their biodistribution by a simple whole animal optical detection, opening new perspectives for cell therapy research and for a variety of diagnosis applications.

Functionalized single-walled carbon nanotubes containing traces of iron as new negative MRI contrast agents for in vivo imaging.

Single-walled carbon nanotubes (SWCNTs) containing traces of iron oxide were functionalized by noncovalent lipid-PEG or covalent carboxylic acid function to supply new efficient MRI contrast agents for in vitro and in vivo applications. Longitudinal (r(1)) and transversal (r(2)) water proton relaxivities were measured at 300 MHz, showing a stronger T(2) feature as an MRI contrast agent (r(2)/r(1) = 190 for CO(2) H functionalisation). The r(2) relaxivity was demonstrated to be correlated to the presence of iron oxide in the SWNT-carboxylic function COOH, in comparison to iron-free ones. Biodistribution studies on mice after a systemic injection showed a negative MRI contrast in liver, suggesting the presence of the nanotubes in this organ until 48 h after i.v. injection. The presence of carbon nanotubes in liver was confirmed after ex vivo carbon extraction. Finally, cytotoxicity studies showed no apparent effect owing to the presence of the carbon nanotubes. The functionalized carbon nanotubes were well tolerated by the animals at the dose of 10 µg g(-1) body weight.

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.

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.

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.

Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand.

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (+/- DOPE) (+/- Lac-10-Chol) that present reproducible absolute entrapment value (1.45 +/- 0.10%), with a size of 109 +/- 7 nm and a slight positive charge (3.77 +/- 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (P > 0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (P < 0.05).

Nanoparticles for imaging and tumor gene delivery.

Molecular imaging of receptors expressed on the surface of tumor cells is becoming a major field of investigation in clinical oncology, especially for the detection of cancer at its earliest stages. Nowadays, MRI, microcomputed tomography (microCT), ultrasound, positron emission tomography (PET), optical coherence tomography (OCT), and other major imaging systems are available to scientists and clinicians. Each technique has advantages and limitations, thus making them complementary. We report herein our investigations on the synthesis and use of new probes for small animal imaging as well as on the preparation and use of targeting complexes to deliver specific gene in tumors cells.