Various lipid anchors on amphiphilic polyoxazolines to reach efficient intracellular delivery.

This work aimed at evaluating the potential of amphiphilic polyoxazolines bearing lipid chain called lipopolyoxazolines to reach efficient intracellular delivery. Four lipid chains: linear saturated, linear unsaturated and two branched one of various length were associated to poly(2-methyl-2-oxazoline) block. The evaluation of their physicochemical features and their impact on cell viability and internalization capacity indicated that the linear saturated gathered the highest cell internalization with a good cell viability. Its intracellular delivery capacity was compared to the PEG reference (DSPE-PEG) after being formulated in liposomes and loaded with fluorescent probe. Both POxylated and PEGylated liposomes showed similar characteristics regarding size distribution, drug loading and cell viability. However, their intracellular delivery was dramatically different, with an improved delivery by 30 folds for the POxylated ones. This significantly better performance highlighted the difficulty of PEGylated liposomes to enter the cells by endocytosis, contrary to POxylated liposomes. This study promotes the value of lipopoly(oxazoline) as a lipopoly(ethylene glycol) alternative for effective intracellular delivery and holds great promises for development of nanoformulations for intravenous administration.

Interactions of amphiphilic polyoxazolines formulated or not in lipid nanocapsules with biological systems: Evaluation from membrane models up to in vivo mice epidermis.

In this study, we evaluated the potential of amphiphilic polyoxazolines (POx) to interact with biological membranes thanks to models of increasing complexity, from a simple lipid bilayer using giant unilamellar vesicles (GUV), to plasma membranes of three different cell types, fibroblasts, keratinocytes and melanocytes, which are found in human skin. Upon assessing an excellent penetration into GUV membranes and cultured cells, we addressed POx's potential to penetrate the murine skin within an in vivo model. Exposure studies were made with native POx and with POx encapsulated within lipid nanocapsules (LNC). Our findings indicate that POx's interactions with membranes tightly depend on the nature of the alkyl chain constituting the POx. Saturated C16POx insert rapidly and efficiently into GUV and plasma membranes, while unsaturated C18:2POx insert to a smaller extent. The high amount of membrane-inserted saturated C16POx impacts cell viability to a greater extent than the unsaturated C18:2POx. The in vivo study, performed on mice, showed an efficient accumulation of both POx types in the stratum corneum barrier, reaching the upper epidermis, independently of POx's degree of saturation. Furthermore, the formulation of POx into lipid nanocapsules allowed delivering an encapsulated molecule, the quercetin, in the upper epidermis layers of murine skin, proving POx's efficacy for topical delivery of active molecules. Overall, POx proved to be an excellent choice for topical delivery, which might in turn offer new possibilities for skin treatments in diseases such as psoriasis or melanomas.

Recent advances and prospects in nano drug delivery systems using lipopolyoxazolines.

Facing the growing demand in nano drug delivery systems (nDDS), hybrid excipients based on natural molecules and well-defined synthetic polymers are intensively investigated. Lipopolyoxazolines (LipoPOx) composed of a polyoxazoline block (POx) and a lipid or lipid-like derivative are detailed in this review. The nature of lipids used, the route to synthesize LipoPOx and their advantages for the formulation of drugs are reported. The place of POx family in nanomedicine is discussed compared to PEG, considered as the gold standard of hydrophilic polymers. LipoPOx nanoformulations including liposomes, mixed micelles, lipid nanocapsules are provided alongside discussion of the nDDS for intravenous or topical administration.

Polyoxazolines based lipid nanocapsules for topical delivery of antioxidants.

Nano-sized lipid formulations offer a great potential for topical delivery of active compounds to treat and prevent human skin damages. Of particular importance is the high loading of hydrophobic molecules, the long-term stability and the auspicious penetration capacity especially reached when using lipid nanocapsules (LNC). Unfortunately, their formation currently relies on a phase inversion process that only operates when using a poly(ethylene glycol) (PEG) based surfactant belonging to the controversial PEG family that was subject of clinical awareness. The present study proposes an alternative to this overused polymer in formulations by designing LNC made of harmless amphiphilic polyoxazolines (POx). Implementing a short sonication step in the process allowed well-defined spherical nanoparticles of ~30 nm to be obtained. The structure of the so called LNC POx was composed of an oily core surrounded by a rigid shell of phospholipids and POx, which ensures a high stability over time, temperature, centrifugation and freezing. Encapsulation of the natural quercetin antioxidant led to a drug loading three times higher than for LNC constituted of PEG (LNC PEG). The antioxidant activity of loaded LNC POx was tested on mice fibroblasts and human keratinocytes after exposure to free radicals from peroxides and UVB irradiation, respectively. The radical scavenging capacity of quercetin loaded in the LNC POx was preserved and even slightly enhanced compared to LNC PEG, highlighting the POx value in nanoformulations.

Combining sol-gel and microfluidics processes for the synthesis of protein-containing hybrid microgels.

Biocompatible chemical cross-linked hybrid polyethylene glycol-based hydrogels were obtained from a sol-gel process using bis-silylated molecular precursors in biocompatible conditions. This soft procedure (pH = 7.4, at 25 °C), allows the production of microgels by microfluidics and easy encapsulation of a model protein (Bovin Serum Albumine, BSA).

Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery.

This study aims to prove the value of the polyoxazolines polymer family as surfactant in formulations for topical application and as an alternative to PEG overuse. The amphiphilic polyoxazolines (POx) were demonstrated to have less impact on cell viability of mice fibroblasts (NIH3T3) than their PEG counterparts. Mixed micelles, made of POx and phosphatidylcholine, were manufactured using thin film and high pressure homogenizer process. The mixed micelles were optimized to produce nanosized vesicles of about 20 nm with a spherical shape and stable over 28 days. The natural lipophilic antioxidant, quercetin, was successfully encapsulated (encapsulation efficiency 94 ±â€¯4% and drug loading 3.6 ±â€¯0.2%) in the mixed micelles with no morphological variation. Once loaded in the formulation, the quercetin impact on cell viability of NIH3T3 was decreased while its antioxidant activity remained unchanged. This work highlights the capacity of amphiphilic POx to create, in association with phospholipids, stable nanoformulations which show promise for topical delivery of antioxidant and ensure skin protection against oxidative stress.

Liposomes, lipid nanocapsules and smartCrystals®: A comparative study for an effective quercetin delivery to the skin.

Quercetin is a flavonoid with strong antioxidant and antiinflammatory activities considered as a potential drug candidate for skin exogenous supplementation. Nevertheless, crude quercetin suffers from poor water solubility and consequently topical inactivity. Therefore, quercetin formulation within a suitable system that overcomes its solubility limitation is a matter of investigation. Three approaches were tested to improve quercetin delivery to skin: liposomes, lipid nanocapsules (LNC) and smartCrystals®. These nanoformulations were compared in terms of average particle size, homogeneity (PDI), quercetin loading and cellular interactions with HaCaT (keratinocytes) and TPH-1 (monocytes) cell lines. Finally, two formulations were selected for testing quercetin delivery to human skin in vivo using stripping test. Different size distribution was obtained with each strategy starting from 26 nm with quercetin LNC, 179 nm with liposomes to 295 nm with quercetin smartCrystals®. The drug loading varied with each formulation from 0.56 mg/ml with liposomes, 10.8 mg/ml with LNC to 14.4 mg/ml with smartCrystals®. No toxicity was observed in HaCaT cells with quercetin and free radical scavenging ability was established at 5 µg/ml. The safety of quercetin at 5 µg/ml was further confirmed on THP-1 cells with efficient free radical scavenging ability. Finally, skin penetration evidenced different behavior between the two selected forms (LNC and SmartCrystals®), which could lead to different promising strategies for skin protection. On one side, quercetin smartCrystals® seems to enable the superficial deposition of quercetin on top of the skin, which presents a good strategy for a quercetin-based sunscreen product. On the other side, LNC seems to allow quercetin delivery to viable epidermis that holds the promise for skin inflammatory disorders such as psoriasis.

Experimental separation steps influence the protein content of corona around mesoporous silica nanoparticles.

In order to direct nanocarriers to their targets efficiently, we have to understand the interactions occurring at the nano-bio interface between nanocarriers and human proteins, which forms the layer called the corona. However, experiments aiming to identify and quantify the proteins in the corona, especially critical steps in the separation of nanoparticles from biological media may affect the corona composition. Here, we used nano-LC MS/MS to compare the protein corona contents obtained after using two different separation methods. We showed that applying centrifugation versus magnetization to isolate nanoparticles surrounded by a corona resulted in protein loss and a reshuffling of their respective abundances.

Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide.

Quercetin is a plant flavonoid with strong antioxidant and antiinflammatory properties interesting for skin protection. However, its poor water solubility limits its penetration and so its efficiency on skin. For this purpose, quercetin lipid nanocapsules were formulated implementing phase inversion technique wherein several modifications were introduced to enhance quercetin loading. Quercetin lipid nanocapsules were formulated with two particle size range, (50nm and 20nm) allowing a drug loading of 18.6 and 32mM respectively. The successful encapsulation of quercetin within lipid nanocapsules increased its apparent water solubility by more than 5000 fold (from 0.5µg/ml to about 5mg/ml). The physicochemical properties of these formulations such as surface charge, stability and morphology were characterized. Lipid nanocapsules had spherical shape and were stable for 28days at 25°C. Quercetin release from lipid nanocapsules was studied and revealed a prolonged release kinetics during 24h. Using DPPH assay, we demonstrated that the formulation process of lipid nanocapsules did not modify the antioxidant activity of quercetin in vitro (92.3%). With the goal of a future dermal application, quercetin lipid nanocapsules were applied to THP-1 monocytes and proved the cellular safety of the formulation up to 2µg/ml of quercetin. Finally, formulated quercetin was as efficient as the crude form in the protection of THP-1 cells from oxidative stress by exogenous hydrogen peroxide. With its lipophilic nature and occlusive effect on skin, lipid nanocapsules present a promising strategy to deliver quercetin to skin tissue and can be of value for other poorly water soluble drug candidates.

Quercetin topical application, from conventional dosage forms to nanodosage forms.

Skin is a multifunctional organ with activities in protection, metabolism and regulation. Skin is in a continuous exposure to oxidizing agents and inflammogens from the sun and from the contact with the environment. These agents may overload the skin auto-defense capacity. To strengthen skin defense mechanisms against oxidation and inflammation, supplementation of exogenous antioxidants is a promising strategy. Quercetin is a flavonoid with very pronounced effective antioxidant and antiinflammatory activities, and thus a candidate of first choice for such skin supplementation. Quercetin showed interesting actions in cellular and animal based models, ranging from protecting cells from UV irradiation to support skin regeneration in wound healing. However, due to its poor solubility, quercetin has limited skin penetration ability, and various formulation approaches were taken to increase its dermal penetration. In this article, the quercetin antioxidant and antiinflammatory activities in wound healing and supporting skin against aging are discussed in detail. In addition, quercetin topical formulations from conventional emulsions to novel nanoformulations in terms of skin penetration enhancement are also presented. This article gives a comprehensive review of quercetin for topical application from biological effects to pharmaceutical formulation design for the last 25 years of research.

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth.

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm-wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50µm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors.

[Current uses and indications for indocyanine green angiography]. / Intérêt et indications actuelles de l'angiographie au vert d'indocyanine (ICG).

A full interpretation of indocyanine green angiography images involves not only optical issues but also pharmacokinetic and biochemical aspects. These issues may involve biochemical changes in the fluorescence yield and the affinity of the molecule for lipoproteins and phospholipids. For age related macular degeneration (AMD), the advent of photodynamic therapy and especially anti-VEGF drugs has increased the use of OCT in assessing treatment response and guiding retreatment. The ease and advantages of OCT have become increasingly associated with a decreasing interest in ICG angiography, which is becoming less well suited for the current management of AMD. An aging population, the efficacy of anti-VEGF drugs and the relative rarity of polypoidal choroidal vasculopathy (PCV) in Europe are factors contributing to our proportional increase in AMD patients. However, aside from AMD, the indications for ICG angiography remain little changed over the last decade: it remains important in diagnosing PCV and choroidal hemangiomas, since their prognosis and treatment are specific. Similarly, for certain inflammatory conditions such as Multiple Evanescent White Dot Syndrome (MEWDS) or Birdshot chorioretinitis, the value of ICG angiography remains significant. In addition, for the treatment of chronic Central Serous Chorioretinopathy, ICG angiography helps to find sites of leakage which otherwise might have been missed. The ICG angiographic appearance in this setting may also have prognostic value. Although the indications for ICG angiography are currently decreasing for AMD, these other conditions represent a large enough number of patients to justify the continued use of this original test, which remains complementary to other chorioretinal imaging techniques.

Density functional theory-based conformational analysis of a phospholipid molecule (dimyristoyl phosphatidylcholine).

The conformational space of the dimyristoyl phosphatidylcholine (DMPC) molecule has been studied using density functional theory (DFT), augmented with a damped empirical dispersion energy term (DFT-D). Fourteen ground-state isomers have been found with total energies within less than 1 kcal/mol. Despite differences in combinations of their torsion angles, all these conformers share a common geometric profile, which includes a balance of attractive, repulsive, and constraint forces between and within specific groups of atoms. The definition of this profile fits with most of the structural characteristics deduced from measured NMR properties of DMPC solutions. The calculated vibrational spectrum of the molecule is in good agreement with experimental data obtained for DMPC bilayers. These results support the idea that DMPC molecules preserve their individual molecular structures in the various assemblies.

Pillaring effects in macroporous carrageenan-silica composite microspheres.

The impregnation of a carrageenan gel by a silica sol is an efficient method to form a composite material which can be conveniently activated by CO2 supercritical drying. The textural properties of the solids have been characterized by nitrogen adsorption-desorption at 77 K and their composition by thermogravimetric analysis and EDX microprobe. Morphology was examined by SEM. The silica-carrageenan composites present an open macroporous structure. Silica particles retained inside the gel behaved as pillars between the polysaccharide fibrils and form a stick-and-ball network. The stiffening of the carrageenan gel by silica prevented its shrinkage upon drying. The nature of the alkali cations affected the retention of silica particles inside the gel. In the absence of silica, carrageenan fibrils rearrange under supercritical drying and form an aerogel with cavities in the mesopore range.

Fluorescence imaging method for in vivo pH monitoring during liposomes uptake in rat liver using a pH-sensitive fluorescent dye.

Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6-carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.

Inclusion of ibuprofen in mesoporous templated silica: drug loading and release property.

The aim of this study is to determine the feasibility of loading biologically active molecules into templated mesoporous silica (MCM 41). This material shows an important mesoporosity associated to hexagonally organized channels, a narrow pore size distribution and a large surface area. Ibuprofen was selected as a model molecule since it is a well documented and much used anti-inflammatory drug. Furthermore, it has a lipophilic character and its molecular size is suitable for inclusion within the mesopores of the MCM 41 material. In order to load ibuprofen within the mesopores, adsorption experiments using various solvents or successive impregnations with solutions of ibuprofen in ethanol were performed. At each step of the loading process, the pore filling was characterized by nitrogen adsorption experiments and by X-ray diffraction. The impregnation procedure results in a significant improvement of the amount of ibuprofen loaded into MCM 41. The in vitro drug release was investigated with simulated biological fluids (gastric and intestinal). Hundred percent release is observed at the end of the in vitro experiment.

Preparation and characterization of siliceous material using liposomes as template.

Material synthesis using unilamellar liposomes with a high sol-gel temperature transition phase as a template leads to a new silica material.

Study of platelet behavior in vivo after endothelial stimulation with laser irradiation using fluorescence intravital videomicroscopy and PEGylated liposome staining.

Platelets contain an array of potent proinflammatory mediators, and therefore they are regarded as mediator and effector cells in inflammation. Knowing the role of platelets during these processes is crucial and the analysis of their behavior in situ and the associated mechanisms is consequently particularly important. However, conventional in vitro staining techniques induce modification of the characteristics of platelets. This study aimed to evaluate platelet behavior in vivo after endothelial stimulation (without endothelial denudation or exposure of basal lamina and/or collagen) with an argon laser, using video intravital microscopy in combination with a new an innovative platelet staining technique based on polyethyleneglycol (PEG) liposomes. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters. Platelets were stained by 5,6-CF-encapsulated PEGylated liposomes injected intravenously. The skin microcirculation was observed with an intravital microscope (using x25, x40, and x80 magnifications) fitted with a xenon light source, an epifluorescence assembly, and an ultra-high sensitivity video camera for fluorescence imaging. Platelet activation without endothelial denudation or exposure of basal lamina and/or collagen was obtained with an argon laser emitting at 514.5 nm with the following parameters: 20 mW, 300 ms, 120 J/cm(2). The 80-microm laser beam was focused on a vessel and its position was controlled with the microscope. Thanks to the spatial resolution of the intravital microscopic imaging system, the platelets were seen rolling individually on the endothelium. After laser stimulation, platelets were activated and three phases were observed: recruitment, adhesion and detachment. The observation of these three phases was time dependent and the kinetics of the process were quantified. The recruitment reached a maximum after 90 +/- 20 s. The adhesion phase lasted for 110 +/- 25 s. At last, detachment of all platelets was observed. This detachment started 200 +/- 20 s after irradiation and was completed in less than 2 min. This study confirms that laser irradiation used with optimal parameters can induce platelet activation without thrombus formation. Platelets can adhere only transiently on stimulated endothelium. This phenomenon may therefore represent a defense mechanism, by which platelets would accumulate in the vicinity of an injury, making them available for immediate response. At last, this study has clearly demonstrated the advantages of our new and innovative platelet staining method using PEGylated liposomes, which are (i) in situ labeling, (ii) use of a hydrophilic marker located in an aqueous compartment within the platelet, and (iii) labeling of platelets allowing observation during the whole experiment.

On-line direct determination of the second virial coefficient of a natural polysaccharide using size-exclusion chromatography and multi-angle laser light scattering.

By combining a size-exclusion chromatographic (SEC) separation and an on-line multi-angle light scattering (MALLS) analysis, we have elaborated an original methodology permitting on-line direct determination of the second virial coefficient of molar mass fractions of polydisperse polysaccharides. By assimilating the SEC-MALLS data to a batch mode acquisition, we have obtained on-line the complete Zimm plot of the eluted fractions, leading to knowledge of their weight-average molar mass Mw, radius of gyration r(g) and second virial coefficient A2. Our methodology was successfully applied to a iota carrageenan sample in LiCl 100 mM, EDTA 1 g/l.

In vivo behaviour of long-circulating liposomes in blood vessels in hamster inflammation and septic shock models-use of intravital fluorescence microscopy.

This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5 and 6-CF-encapsulated polyethylene glycolated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope (using x40, x80 magnification) fitted with a Xenon light source and an epifluorescence assembly (excitation, 470 nm, FWHM 40 nm; emission, 540 nm, FWHM 40 nm). An ultra-high sensitivity videocamera mounted on the microscope projected the image onto a monitor, and the images (720 x 576 pixels) were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using this model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 h. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up, liposomes. This observation is in accordance with previous in vitro studies.