Tunable pH Sensitive Lipoplexes.

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 H+-ATPase inhibitor.

Lipids for Nucleic Acid Delivery: Cationic or Neutral Lipoplexes, Synthesis, and Particle Formation.

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.

Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics.

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.

Novel Perfluorinated Triblock Amphiphilic Copolymers for Lipid-Shelled Microbubble Stabilization.

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.

Cyanine derivative as a suitable marker for thermosensitive in situ gelling delivery systems: In vitro and in vivo validation of a sustained buccal drug delivery.

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.

Cationic microbubbles and antibiotic-free miniplasmid for sustained ultrasound-mediated transgene expression in liver.

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.

Characterization of Positively Charged Lipid Shell Microbubbles with Tunable Resistive Pulse Sensing (TRPS) Method: A Technical Note.

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.

Controlling aminosilane layer thickness to extend the plasma half-life of stealth persistent luminescence nanoparticles in vivo.

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.

Fine tuning of mixed ionic and hydrogen bond interactions for plasmid delivery using lipoplexes.

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.

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.

Cationic lipid nanocarriers activate Toll-like receptor 2 and NLRP3 inflammasome pathways.

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-κB activation, and the production of TNF-α, IL-1ß, IL-6 and IFN-γ 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.

Lipidic spherulites: formulation optimisation by paired optical and cryoelectron microscopy.

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.

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.

Lipids for nucleic acid delivery: synthesis and particle formation.

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 wish to 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.

Xyloglucan-derivatized films for the culture of adherent cells and their thermocontrolled detachment: a promising alternative to cells sensitive to protease treatment.

By taking advantage of a natural and abundant polymer as well as a straightforward film formation technique, this paper focuses on the conception and use of a new alternative tool for thermo-controlled cell detachment. Thermoresponsive xyloglucan was produced after partial galactose removal by a 24 h reaction with ß-galactosidase. The obtained polymer (T24) was then activated by reaction with 4-nitrophenyl chloroformate (NPC) in order to graft a cyclic peptide presenting an arginine-glycine-aspartic acid (RGD) motif. The effect of RGD grafting on the sol-gel transition temperature of T24 is evaluated by rheological measurements. Solvent-casted films of T24-RGD successfully promoted cell adhesion, proliferation, and thermo-controlled detachment. The presented approach is a new alternative for cells sensitive to the proteolytic treatment routinely used for cell detachment. Because the RGD sequence used herein is widely recognized by different cell types, this protocol may be extended to other cells. Besides, the presented chemical route can be applied to different peptide sequences.

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.

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.

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.