To provide long circulating nanoparticles able to carry a gene to tumor cells, we have designed anionic pegylated lipoplexes which are pH sensitive. The reduction of positive charges in nucleic acid carriers allows reducing the elimination rate, increasing circulation time in the blood, leading to improved tumor accumulation of lipid nanoparticles. Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. The neutralization of the particle surface charge as a function of the pH was monitored by dynamic light scattering in order to determine the ratio between anionic and cationic lipids that would give pH -sensitive complexes. This ratio has been optimized to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to Picogreen. The transfection efficiency and pH -sensitive property of these formulations were shown in vitro using bafilomycin, a vacuolar Fit ATPase inhibitor.

Lipidic vesicles have been extensively studied for their capacity to condensate and deliver nucleic acids to the cells. Many different amphiphilic lipidic structures have been proposed each of them bringing some advances in nonviral gene transfection. The ionic or neutral nature of the lipids induces tremendous differences in the behavior of the corresponding liposomes, from the complexation of nucleic acid to the delivery to the cell. An efficient delivery in vitro or in vivo also depends closely on the structure of the lipids and very often, efficient liposomes in vitro have been found useless for in vivo administration. We describe in this chapter the chemical synthesis of two different lipids, one cationic and the other essentially neutral, and the formulation to obtain liposomes and DNA-liposome complexes. The different ways and tricks for the formulation of the two different structures are especially highlighted.

Lactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.

Amphiphilic triblock (Atri) copolymers made of perfluorinated alkyl chain linked to hydrocarbon chain and methoxy-poly(ethylene glycol) of three different molecular weights were synthesized. In vitro evaluation demonstrated that these new compounds were noncytotoxic. Characterization and interaction of each triblock copolymer with a branched polyamine myristoyl lipid (2-{3[bis-(3-amino-propyl)-amino]-propylamino}- N-ditetradecyl carbamoyl methyl-acetamide, DMAPAP) were studied by the Langmuir film method and thermal analysis. The triblock copolymer/cationic lipids (1:10, w/w) were mixed with perfluorobutane gas to form microbubbles (MBs). The latter were characterized by optical microscopy to get the microbubble size and concentration by densimetry to determine the amount of encapsulated gas and by ultrasound to assess oscillation properties. Atri with the lowest and intermediate weights were shown to interact with the cationic lipid DMAPAP and stabilize the Langmuir film. In that case, monodisperse microbubbles ranging from 2.3 ± 0.1 to 2.8 ± 0.1 μm were obtained. The proportion of encapsulated gas within the MB shell increased up to 3 times after the incorporation of the copolymer with the lowest and intermediate weights. Moreover, the acoustic response of the microbubbles was maintained in the presence of the copolymers.

In the context of increasing liver diseases, no contrast agent is currently available in Europe and the United States to directly assess the liver function. Only neolactosylated human serum albumin is being clinically used in Asia. In order to perform preclinical studies in the context of liver diseases, we conceived a fluorescent lactosylated albumin for the quantification of liver functional cells (l-Cyal). Precise characterization was achieved in order to determine the amounts of lactose and Cyanine 5 (Cy5) coupled to the albumin. In addition, potential aggregation was characterized by asymmetrical flow field-flow fractionation hyphenated to multi-angle light scattering (AF4-MALS). The optimal functionalized albumin exhibited a mass greater than 87 kDa which corresponds to the addition of 34 lactose moieties per protein and 1-2 Cy5 labels. Also, no significant formation of aggregates could be identified due to the modification of the native albumin. In healthy mice, the accumulation of l-Cyal in the liver and its selectivity for hepatocyte cells were shown by optical imaging and flow cytometry. Administration of l-Cyal to mice bearing liver metastases showed a reduced signal in the liver related to a decrease in the number of hepatocytes. The l-Cyal bioimaging contrast agent could be particularly useful for assessing the state of liver related diseases.

Despite the increasing number of clinical trials in gene therapy, no ideal methods still allow non-viral gene transfer in deep tissues such as the liver. We were interested in ultrasound (US)-mediated gene delivery to provide long term liver expression. For this purpose, new positively charged microbubbles were designed and complexed with pFAR4, a highly efficient small length miniplasmid DNA devoid of antibiotic resistance sequence. Sonoporation parameters, such as insonation time, acoustic pressure and duration of plasmid injection were controlled under ultrasound imaging guidance. The optimization of these various parameters was performed by bioluminescence optical imaging of luciferase reporter gene expression in the liver. Mice were injected with 50μg pFAR4-LUC either alone, or complexed with positively charged microbubbles, or co-injected with neutral MicroMarker™ microbubbles, followed by low ultrasound energy application to the liver. Injection of the pFAR4 encoding luciferase alone led to a transient transgene expression that lasted only for two days. The significant luciferase signal obtained with neutral microbubbles decreased over 2days and reached a plateau with a level around 1 log above the signal obtained with pFAR4 alone. With the newly designed positively charged microbubbles, we obtained a much stronger bioluminescence signal which increased over 2days. The 12-fold difference (p<0.05) between MicroMarker™ and our positively charged microbubbles was maintained over a period of 6months. Noteworthy, the positively charged microbubbles led to an improvement of 180-fold (p<0.001) as regard to free pDNA using unfocused ultrasound performed at clinically tolerated ultrasound amplitude. Transient liver damage was observed when using the cationic microbubble-pFAR4 complexes and the optimized sonoporation parameters. Immunohistochemistry analyses were performed to determine the nature of cells transfected. The pFAR4 miniplasmid complexed with cationic microbubbles allowed to transfect mostly hepatocytes compared to its co-injection with MicroMarker™ which transfected more preferentially endothelial cells.

Buccal administration route is a promising way for a large number of drugs exhibiting a low oral bioavailability. The present work describes the formulation and evaluation of a mucoadhesive and thermosensitive in situ gelling delivery system based on poloxamer 407, poloxamer 188 and xanthan gum for buccal drug delivery. First, the mucoadhesion properties were evaluated using a tensile test. The effect of xanthan gum on the mucoadhesion force was demonstrated. Then, to assess the buccal residence time which reflects the mucoadhesion properties, the validation of a fluorescence probe for in vivo optical imaging experiment was conducted. Methyl-Cyanine 5 derivative (Me-Cy5) was used to label the hydrogels, dissolution tests and permeation studies through buccal epithelium cells showed that Me-Cy5 release from hydrogels was mainly due to an erosion mechanism and presented a limited penetration across epithelium cells. These results suggest that, Me-Cy5 is a suitable marker for thermosensitive in situ gelling delivery systems as the probe mostly stays entrapped in the hydrogel and do not cross the epithelial barrier. Buccal residence performance of the hydrogel was evaluated for the first time by non-invasive optical imaging after administration to mice. This technique is an interesting alternative compared to visual observations and sacrifice involved experiments, which could also be exploited to various administration routes.

Ultrasound is widely used in the medical imaging field for diagnostic purposes. Twenty years ago, their use has turned to therapeutic applications for musculoskeletal disorders mainly The invention of gas microbubble ultrasound contrast agent was the trigger of a large number of studies on extra-molecules delivery into cells by coupling ultrasound and gas microbubbles. This technique called sonoporation has the advantage to deliver locally drugs near or loaded inside microbubbles. Gas microbubble is therefore serves as a drug reservoir that could be triggered by ultrasound. In this study, we developed gas microbubbles capable of delivering both a cytotoxic agent as Paclitaxel and siRNA to promote tumour regression. Microbubbles were developed with original histidylated cationic lipids which are pH-sensitive. In vitro measurements have been done to depict the behaviour of these microbubbles in the presence of tumour cells after ultrasound activation. Confocal microscopy and flow cytometry experiments demonstrated that cationic microbubbles were able to complex siRNA. Ultrasound parameters were optimized in vitro for siRNA delivery in 4T1 cells stably expressing luciferase (luc). Gas-filled cationic microbubbles complexed with anti-luc siRNA led to specific inhibition of luciferase expression under specific ultrasound parameters. A proof of principle has been validated in vivo using 4T1 orthotopic murine mammary tumour model. Our siRNA-Paclitaxel formulations were injected either inside the tumour or in the blood circulation via tail vein followed by ultrasound application at tumour site. A significant inhibition of tumor growth were observed in mice treated weekly with these formulations repeated at least three times. In conclusion, we succeeded to produce original gas-filled liposomes made with histidylated cationic liposomes that were ultrasound-sensitive and able to co-deliver efficiently Paclitaxel and siRNA for tumor treatment.

Therapeutics and diagnostics both initiated the development and rational design of nanoparticles intended for biomedical applications. Yet, the fate of these nanosystems in vivo is hardly manageable and generally results in their rapid uptake by the mononuclear phagocyte system, i.e. liver and spleen. To overcome this essential limitation, efforts have been made to understand the influence of physico-chemical parameters on the behaviour of nanoparticles in vivo and on their ability to be uptaken by phagocytic cells. Notably, polyethylene glycol grafting and precise control of its density have not only been shown to prevent protein adsorption on the surface of nanoparticles, but also to significantly reduce macrophage uptake in vitro. In this article, we suggest the use of persistent luminescence to study the influence of another parameter, aminosilane layer thickness, on both in vitro protein adsorption and in vivo biodistribution of stealth persistent nanophosphors.

Microbubbles are polydisperse microparticles. Their size distribution cannot be accurately measured from the current methods used, such as optical microscopy, electrical sensing or light scattering. Indeed, these techniques present some limitations when applied to microbubbles, which prompted us to investigate the use of an alternative technique: tunable resistive pulse sensing (TRPS). This technique is based on the principle of the Coulter counter with the advantage of being more flexible compared to other methods using this principle, since the flow rate, the potential difference and the pore size can be modulated. The main limitation of TRPS is that more than one size of nanopore membrane is required to obtain the full size distribution of polydisperse microparticles. To evaluate this technique, the concentration and the size distribution of positively charged microbubbles were studied using TRPS and compared to data obtained using optical microscopy. We describe herein the parameters required for the accurate measurement of microbubble concentration and size distribution by TRPS and present a statistical comparison of the data obtained by TRPS and optical microscopy.

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.

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.

Magnetic resonance imaging is an excellent technique to achieve anatomical details and highly resolved images. The search for efficient contrast agents to increase the signal to background ratio led us to evaluate paramagnetic spherulites as potential Magnetic Resonance Imaging (MRI) contrast agents. Spherulites are supramolecular assemblies, made of lipidic concentric multilayers, able to encapsulate with high efficiency soluble macromolecules. Despite their highly interesting structure, spherulites have never been proposed as imaging agents. We proposed here three approaches to render spherulites paramagnetic: encapsulating a soluble contrastophore, inserting a lipidic contrastophore derivative or grafting a soluble contrastophore at the surface of the spherulites. Following similar strategies, liposomes were prepared for comparison. The conservation of the spherulite structure, throughout these three strategies, was shown by cryoelectron microscopy and small angle light scattering. The effect of the paramagnetic spherulites was studied by magnetic resonance imaging at different magnetic fields. The results showed that insertion of a contrastophore lipidic derivative into spherulite bilayers and grafting a contrastophore at the surface of the spherulites were the two strategies which led to the highest MRI contrast improvement.

Non viral gene transfection has been mostly reached via cationic polymer and lipid, required for DNA complexation and cell internalisation. However, cationic charges often induce cytotoxicity and limit the efficacy of the lipoplexes in vivo due to their fast elimination from the blood stream. Few years ago, we had developed noncationic lipid interacting with DNA via hydrogen bond interactions. To take advantage of both the internalisation efficacy of cationic complexes and the higher DNA release efficacy of non cationic lipids, we chose to mix both ionic and hydrogen bond interactions within one lipoplex. The idea behind this strategy would be to reduce the overall charge while maintaining a high level of transfection. Four mixed formulations of cationic lipid and thiourea lipid were prepared. We found that decreasing ionic interactions and increasing hydrogen bond interactions improved cationic lipoplexes properties. Indeed, we showed that replacement of net positive charges by hydrogen bond interactions with DNA phosphates led to efficient lipoplexes for in vitro DNA transfection at lower cationic charge content, which consequently reduced lipoplex cytotoxicity.

Recent breakthroughs in the rational development of multifunctional nanocarriers have highlightened the advantage of combining the complementary forces of several imaging modalities into one single nanotool fully dedicated to the biomedical field and diagnosis applications. A novel multimodal optical‐magnetic resonance imaging nanoprobe is introduced. Designed on the basis of a spinel zinc gallate structure doped with trivalent chromium and gadolinium, this nanocrystal bears the ability to serve as both a highly sensitive persistent luminescence nanoprobe for optical imaging, and a negative contrast agent for highly resolved magnetic resonance imaging (MRI). Additional proof is given that surface coverage can be modified in order to obtain stealth nanoparticles highly suitable for real‐time in vivo application in mice, showing delayed reticulo‐endothelial uptake and longer circulation time after systemic injection.

Objective of this study was to assess the various steps leading to spherulite obtention by means of optical and cryoelectron microscopy. The formulation, resting and hydration steps were optimised. Green-based process and organic-based process were compared. It was found that spherulites could be obtained only when two key steps were followed: a prior resting phase of excipients and the shearing stress of the hydrated excipients. Moreover, the new formulation under study formed spherulites in the 100 200 nm range, which is smaller than previously reported spherulites. Such laboratory scale optimised process led the integration of spherulites in a larger number of prospective studies. Indeed, we finally showed that the encapsulated payload of a hydrophobic compound, such as the anti-angiogenic agent fisetin, was increased to a much higher degree than with a liposomal encapsulation. (C) 2013 Elsevier B.V. All rights reserved.

Une neurotoxine, inhibant la synthèse protéique dans des cellules en culture, a été isolée d'écorces de racines de Cnestis glabra (Connaracées) avec un rendement de l'ordre de 0.4 pour cent. Le procédé de purification utilisé comporte 5 étapes : filtration sur une couche de charbon actif, traitement par l'acétate neutre de plomb et fractionnements par chromatographies successives sur Dowex 50 × 8 sous forme H+ et NH4+. L'homogénéité de la toxine purifiée a été vérifiée par chromatographies sur couche mince et en phase gazeuse. Son poids moléculaire est inférieur à 500. C'est un composé thermostable, insoluble dans les solvants organiques usuels et qui donne une réaction positive à la ninhydrine. L'hydrolyse acide ne modifie pas son comportement sur un analyseur d'acides aminés. Sa nature possible (acide aminé) est discutée. La toxine est provisoirement appeléc glabrine.A neurotoxic compound, inhibiting protein synthesis in cell culture, was isolated in a yield of about 0.4 per cent from Cnestis glabra root barks (Connaraceae) by a five-step fractionation procedure (filtration on activated charcoal, treatment by neutral lead acetate and fractionations on Dowex 50 × 8 in H+ and NH4+ forms). The purified toxin appeared homogeneous on thin-layer and in gas chromatography. The compound has a low molecular wweight (less than 500). It is heat-stable, insoluble in usual organic solvents and gives a positive reaction with ninhydrin. Acidic hydrolysis does not change its behaviour on an amino acid analyzer. Its possible amino acid nature is discussed. It is temporarily named glabrin.

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 +/- 2.4 nm), 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 (21 mg/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. (C) 2013 Elsevier B. V. All rights reserved.

We provide evidence that cationic lipids, usually considered as a safe alternative to viral vectors as nanocarriers for gene therapy or drug intracellular delivery, do not behave as inert material but do activate cellular signalling pathways implicated in inflammatory reactions. We show here that the cationic lipid RPR206252 induces NF-kappa B activation, and the production of TNF-alpha, IL-1 beta, IL-6 and IFN-gamma by human or mouse macrophage cell lines. Further, we demonstrate that the activation of inflammatory cascades by RPR206252 is dependent on Toll-like receptor 2 (TLR2), the natural sensor of bacterial lipopeptides and NOD-like receptor protein 3 (NLRP3), the major inflammasome component. Our results suggest that cationic lipid nanocarriers because of their ability to stimulate the innate system can be used as a new class of synthetic and safe adjuvant for vaccination.From the Clinical Editor: Cationic lipid nanocarriers are typically considered neutral tools for gene delivery. However, as demonstrated in this study, they possess a clear ability to stimulate the innate immune system, and actually can be used as a new class of synthetic and safe adjuvant for vaccination. (C) 2014 Elsevier Inc. All rights reserved.