LipoParticles: a lipid membrane coating onto polymer particles to enhance the internalization in osteoblast cells.

LipoParticles, core-shell assemblies consisting of a polymer core coated by a lipid membrane, are promising carriers for drug delivery applications with intracellular targets. This is of great interest since it is actually challenging to treat infections involving intracellular bacteria such as bone and joint infections where the bacteria are hidden in osteoblast cells. The present work reports for the first time to the best of our knowledge the proof of enhanced internalization of particles in osteoblast cells thanks to a lipid coating of particles (= LipoParticles). The ca. 300 nm-sized assemblies were elaborated by reorganization of liposomes (composed of DPPC/DPTAP 10/90 mol/mol) onto the surface of poly(lactic-co-glycolic acid) (PLGA) particles, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zetametry. Optimization of these assemblies was also performed by adding poly(ethylene glycol) (PEG) chains on their surface (corresponding to a final formulation of DPPC/DPTAP/DPPE-PEG5000 8/90/2 mol/mol/mol). Interestingly, this provided them colloidal stability after their 20-fold dilution in PBS or cell culture medium, and made possible their freeze-drying without forming aggregates after their re-hydration. Their non-cytotoxicity towards a human osteoblast cell line (MG63) was also demonstrated. The enhanced internalization of LipoParticles in this MG63 cell line, in comparison with PLGA particles, was proven by observations with a confocal laser scanning microscope, as well as by flow cytometry assays. Finally, this efficient internalization of LipoParticles in MG63 cells was confirmed by TEM on ultrathin sections, which also revealed localization close to intracellular Staphylococcus aureus.

Small anticancer drug release by light: Photochemical internalization of porphyrin-ß-cyclodextrin nanoparticles.

Aiming to engineer simple, neutral, strongly amphiphilic photoactive nanoparticles (NPs) to specifically target cancer cell lysosomes for drug transport and light-controlled release, new conjugates of ß-cyclodextrin with highly hydrophobic triphenylporphyrin bearing different alkyl chains, were synthesized. Although differently sized, all conjugates self-assemble into ~60 nm NPs in water and display similar photoactivity. The NPs target selectively the lysosomes of breast adenocarcinoma MCF-7 cells, embedding in vesicular membranes, as experiments with model liposomes indicate. Either empty or drug-loaded, the NPs lack dark toxicity for 48 h. They bind with differently structured anticancer drugs tamoxifen and gemcitabine as its N-adamantyl derivative. Red light irradiation of cells incubated with drug-loaded NPs results in major reduction of viability (>85 %) for 48 h displaying significant synergy of photo-chemotoxicity, as opposed to empty NPs, and to loaded non-irradiated NPs, in manifestation of photochemical internalization (PCI). Our approach expands the field of PCI into different small molecule chemotherapeutics.

LipoParticles: Lipid-Coated PLA Nanoparticles Enhanced In Vitro mRNA Transfection Compared to Liposomes.

The approval of two mRNA vaccines as urgent prophylactic treatments against Covid-19 made them a realistic alternative to conventional vaccination methods. However, naked mRNA is rapidly degraded by the body and cannot effectively penetrate cells. Vectors capable of addressing these issues while allowing endosomal escape are therefore needed. To date, the most widely used vectors for this purpose have been lipid-based vectors. Thus, we have designed an innovative vector called LipoParticles (LP) consisting of poly(lactic) acid (PLA) nanoparticles coated with a 15/85 mol/mol DSPC/DOTAP lipid membrane. An in vitro investigation was carried out to examine whether the incorporation of a solid core offered added value compared to liposomes alone. To that end, a formulation strategy that we have named particulate layer-by-layer (pLbL) was used. This method permitted the adsorption of nucleic acids on the surface of LP (mainly by means of electrostatic interactions through the addition of LAH4-L1 peptide), allowing both cellular penetration and endosomal escape. After a thorough characterization of size, size distribution, and surface charge- and a complexation assessment of each vector-their transfection capacity and cytotoxicity (on antigenic presenting cells, namely DC2.4, and epithelial HeLa cells) were compared. LP have been shown to be significantly better transfecting agents than liposomes through pLbL formulation on both HeLa and DC 2.4 cells. These data illustrate the added value of a solid particulate core inside a lipid membrane, which is expected to rigidify the final assemblies and makes them less prone to early loss of mRNA. In addition, this assembly promoted not only efficient delivery of mRNA, but also of plasmid DNA, making it a versatile nucleic acid carrier that could be used for various vaccine applications. Finally, if the addition of the LAH4-L1 peptide systematically leads to toxicity of the pLbL formulation on DC 2.4 cells, the optimization of the nucleic acid/LAH4-L1 peptide mass ratio becomes an interesting strategy-essentially reducing the peptide intake to limit its cytotoxicity while maintaining a relevant transfection efficiency.

Drug-Loaded Lipid-Coated Hybrid Organic-Inorganic "Stealth" Nanoparticles for Cancer Therapy.

Hybrid porous nanoscale metal organic frameworks (nanoMOFs) made of iron trimesate are attracting increasing interest as drug carriers, due to their high drug loading capacity, biodegradability, and biocompatibility. NanoMOF surface modification to prevent clearance by the innate immune system remains still challenging in reason of their high porosity and biodegradable character. Herein, FDA-approved lipids and poly(ethylene glycol) (PEG)-lipid conjugates were used to engineer the surface of nanoMOFs by a rapid and convenient solvent-exchange deposition method. The resulting lipid-coated nanoMOFs were extensively characterized. For the first time, we show that nanoMOF surface modification with lipids affords a better control over drug release and their degradation in biological media. Moreover, when loaded with the anticancer drug Gem-MP (Gemcitabine-monophosphate), iron trimesate nanoMOFs acted as "Trojan horses" carrying the drug inside cancer cells to eradicate them. Most interestingly, the PEG-coated nanoMOFs escaped the capture by macrophages. In a nutshell, versatile PEG-based lipid shells control cell interactions and open perspectives for drug targeting.

Reducing-End Functionalization of 2,5-Anhydro-d-mannofuranose-Linked Chitooligosaccharides by Dioxyamine: Synthesis and Characterization.

The nitrous acid depolymerization of chitosan enables the synthesis of singular chitosan oligosaccharides (COS) since their reducing-end unit is composed of 2,5-anhydro-d-mannofuranose (amf). In the present study, we describe a chemical method for the reducing-end conjugation of COS-amf by the commercially available dioxyamine O,O'-1,3-propanediylbishydroxylamine in high mass yields. The chemical structure of resulting dioxyamine-linked COS-amf synthesized by both oximation and reductive amination ways were fully characterized by 1H- and 13C-NMR spectroscopies and MALDI-TOF mass spectrometry. The coupling of chemically attractive linkers such as dioxyamines at the reducing end of COS-amf forms a relevant strategy for the development of advanced functional COS-based conjugates.

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry.

MALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an antioxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any antioxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain-chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pKa of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains.

Innovative particle standards and long-lived imaging for 2D and 3D dSTORM.

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology.

We report the site-specific and covalent bioconjugation of fluorescent polymer chains to proteins in live cells using the HaloTag technology. Polymer chains bearing a Halo-ligand precisely located at their α-chain-end were synthesized in a controlled manner owing to the RAFT polymerization process. They were labeled in lateral position by several organic fluorophores such as AlexaFluor 647. The resulting Halo-ligand polymer probe was finally shown to selectively recognize and label HaloTag proteins present at the membrane of live cells using confocal fluorescence microscopy. Such a polymer bioconjugation approach holds great promises for various applications ranging from cell imaging to cell surface functionalization.

Comb-like dextran copolymers: A versatile strategy to coat highly porous MOF nanoparticles with a PEG shell.

Nanoparticles made of metal-organic frameworks (nanoMOFs) are becoming of increasing interest as drug carriers. However, engineered coatings such as poly(ethylene glycol) (PEG) based ones are required to prevent nanoMOFs recognition and clearance by the innate immune system, a prerequisite for biomedical applications. This still presents an important challenge due to the highly porous structure and degradability of nanoMOFs. We provide here a proof of concept that the surface of iron-based nanoMOFs can be functionalized in a rapid, organic solvent-free and non-covalent manner using a novel family of comb-like copolymers made of dextran (DEX) grafted with both PEG and alendronate (ALN) moieties, which are iron complexing groups to anchor to the nanoMOFs surface. We describe the synthesis of DEX-ALN-PEG copolymers by click chemistry, with control of both the amount of PEG and ALN moieties. Stable DEX-ALN-PEG coatings substantially decreased their internalization by macrophages in vitro, providing new perspectives for biomedical applications.

Reducing-end "clickable" functionalizations of chitosan oligomers for the synthesis of chitosan-based diblock copolymers.

Chitooligosaccharides (COS) produced by nitrous acid depolymerization of chitosan are unique chitosan oligomers due to the presence of the 2,5-anhydro-d-mannofuranose (amf) unit at their reducing end. In this work, we focused on the reductive amination and the oximation of the amf aldehyde group towards various functionalized anilines, hydrazides and O-hydroxylamines. The aim of this work was to synthesize new COS-based building blocks functionalized at their reducing end by different "clickable" chemical groups such as alkene, alkyne, azide, hydrazide and thiol. Targeted functionalized COS were synthesized in excellent mass yields and fully characterized by NMR spectroscopy and MALDI-TOF mass spectrometry. Our results showed these functionalizations are quantitative, versatile and can be easily performed in mild reaction conditions. Finally, these COS-based building blocks could be useful intermediates for the development of advanced functional COS-based conjugates, as illustrated in this work by the synthesis of new COS-poly(ethylene glycol) (PEG) diblock copolymers.

Tunable morphology of lipid/chitosan particle assemblies.

Lipid/chitosan (CS) particle assemblies have recently been developed as new promising carriers for drug delivery applications. The present work reports for the first time the formation of such assemblies by a simple spontaneous adsorption of lipid membranes onto the CS particle surfaces. As shown by dynamic light scattering (DLS) measurements, final non-aggregated assemblies with relatively satisfactory size distributions were obtained by using this process. Furthermore, a particular attention has been paid herein to the effect of the initial morphology of lipid membranes (i.e., vesicular or discoidal) on the resulting characteristics of assemblies. To this end, each one of these membranes was mixed with CS particles, and the obtained assemblies were observed by transmission electron microscopy (TEM). According to these observations, the vesicular lipid membranes seem to wrap mostly CS particles. In contrast, lipid discs are not reorganized onto the particle surface but would rather be stacked onto the CS particle.

A close collaboration of chitosan with lipid colloidal carriers for drug delivery applications.

Chitosan and lipid colloids have separately shown a growing interest in the field of drug delivery applications. Their success is mainly due to their interesting physicochemical behaviors, as well as their biological properties such as bioactivity and biocompatibility. While chitosan is a well-known cationic polysaccharide with the ability to strongly interact with drugs and biological matrices through mainly electrostatic interactions, lipid colloids are carriers particularly recognized for the drug vectorization. In recent years, the combination of both entities has been considered because it offers new systems which gather the advantages of each of them to efficiently deliver various types of bioactive species. The purpose of this review is to describe these associations between chemically-unmodified chitosan chains (solubilized or dispersed) and lipid colloids (as nanoparticles or organized in lipid layers), as well as their potential in the drug delivery area so far. Three assemblies have mainly been reported in the literature: i) lipid nanoparticles (solid lipid nanoparticles or nanostructured lipid carriers) coated with chitosan chains, ii) lipid vesicles covered with chitosan chains, and iii) chitosan chains structured in nanoparticles with a lipid coating. Their elaboration processes, their physicochemical characterization, and their biological studies are detailed and discussed herein. The different bioactive species (drugs and bio(macro)molecules) incorporated in these assemblies, their maximal incorporation efficiency, and their loading capacity are also presented. This review reveals the versatility of these assemblies. Depending on the organization of lipids (i.e., nanoparticles or vesicles) and the state of polymer chains (i.e., solubilized or dispersed under the form of nanoparticles), a large variety of drugs can be successfully incorporated, and various routes of administration can be considered.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

Toward an optimized treatment of intracellular bacterial infections: input of nanoparticulate drug delivery systems.

Intracellular pathogenic bacteria can lead to some of the most life-threatening infections. By evolving a number of ingenious mechanisms, these bacteria have the ability to invade, colonize and survive in the host cells in active or latent forms over prolonged period of time. A variety of nanoparticulate systems have been developed to optimize the delivery of antibiotics. Main advantages of nanoparticulate systems as compared with free drugs are an efficient drug encapsulation, protection from inactivation, targeting infection sites and the possibility to deliver drugs by overcoming cellular barriers. Nevertheless, despite the great progresses in treating intracellular infections using nanoparticulate carriers, some challenges still remain, such as targeting cellular subcompartments with bacteria and delivering synergistic drug combinations. Engineered nanoparticles should allow controlling drug release both inside cells and within the extracellular space before reaching the target cells.

Liposome-loaded chitosan physical hydrogel: toward a promising delayed-release biosystem.

This work deals with the elaboration of an original biosystem in view of its application as drug delayed-release device in biomedical area. This innovative "hybrid" system is composed of phosphatidylcholine liposomes entrapped within a chitosan physical hydrogel (only constituted of polymer and water). To this end, pre-formed liposomes were suspended into chitosan solutions, and the polymer gelation process was subsequently carried out following particular experimental conditions. This liposome incorporation did absolutely not prevent the gel formation as shown by rheological properties of the resulting tridimensional matrix. The presence of liposomes within the hydrogel was confirmed by fluorescence and cryo-scanning electron microscopies. Then, the expected concept of delayed-release of this "hybrid" system was proved using a model water soluble molecule (carboxyfluorescein, CF) encapsulated in liposomes, themselves incorporated into the chitosan hydrogel. The CF release was assayed after repeated and intensive washings of hydrogels, and was found to be higher in the CF-in-hydrogel systems in comparison with the CF-in-liposomes-in-hydrogel ones, demonstrating a CF delayed-release thanks to lipid vesicles.

Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer.

Nowadays, the easy access of tetra-N-acetyl-chitopentaose and its counterparts is highly interesting since such chemical compounds are precursors of biological signal molecules with a strong agro-economic impact. The chemical synthesis of tetra-N-acetyl-chitopentaose by controlled N-acetylation of the glucosamine pentamer hydrochloride under mild conditions is described herein. A systematic study on the influence of the different parameters involved in this reaction, such as the solvent, the acetylating agent, and the base used for the deprotonation of ammonium groups of the starting material was carried out. The characterization of final reaction products by HPLC and MALDI-TOF mass spectrometry showed that each of these parameters affects differently the acetylation reaction. Whereas the solvent plays an important role in the N- or O-acetylation selectivity, the acetylating agent and the base were found to influence both the degree of N-acetylation and the distribution of the partially N-acetylated derivatives in the product mixtures. Based on these results, optimized reaction conditions have been established allowing tetra-N-acetyl-chitopentaose to be synthesized in a one-pot deprotonation/N-acetylation of the glucosamine pentamer hydrochloride in a moderate yield (ca 30%).

Ring-opening polymerization with Zn(C6F5)2-based Lewis pairs: original and efficient approach to cyclic polyesters.

Dual systems combining Zn(C6F5)2 with an organic base (an amine or a phosphine) promote the controlled ring-opening polymerization of lactide and ε-caprolactone. The Lewis pairs cooperate to activate the monomers, affording well-defined high molecular weight cyclic polyesters. Efficient chain-extension gives access to cyclic block copolymers.

Shear thinning three-dimensional colloidal assemblies of chitosan and poly(lactic acid) nanoparticles.

In this study, new materials capable of reversible self-assembly, based on concentrated negatively charged poly(lactic acid) nanoparticles and chitosan, a natural polycationic polymer, were successfully fabricated. Electrostatic interactions between oppositely charged components along with weaker interactions led to the formation of a 3D network. The resulting macroscopic assemblies were characterized by dynamic mechanical measurements, and the influences of various parameters such as chitosan/poly(lactic acid) weight ratio, duration and temperature of the mixture, and molecular weight or chitosan degree of acetylation were studied. Our results showed that the mechanical properties of assemblies were highly dependent on the nanoparticle solid content and chitosan/nanoparticle ratio. In particular, at an optimum weight ratio the colloidal assemblies exhibited remarkable high elastic moduli (about 300 kPa) for a particle solid content of 18% w/w. Thanks to the weak and reversible nature of the interactions, these materials exhibited shear thinning properties, and could instantly recover their cohesiveness at rest. The mode of interactions between PLA particles and chitosan was shown to be in part due to electrostatic interactions, but the cross-linking of chitosan-covered particles was attributed to hydrogen bonding. These materials could be envisaged as good candidates for injectable scaffolds for tissue engineering, taking advantage of the biocompatibility and bioactivity of both components. However, some issues concerning temperature stability must be resolved before applying these colloidal assemblies to cell growth in physiological conditions.

Synthesis of water-soluble and water-insoluble amphiphilic derivatives of dextran in organic medium.

Hydrophobically modified dextrans were prepared by reacting native polysaccharide with 1,2-epoxydodecane in dimethylsulfoxide. Epoxide oligomerization was shown to occur as a secondary reaction when hydroxide ions were used as base catalysts. By adjusting the amount of epoxide in the feed, dextran derivatives with degrees of substitution (DS) between 0% and 164% were obtained. Polymers with DS above 100% were readily soluble in organic solvents like tetrahydrofuran, dioxane and water-saturated chloroform and dichloromethane. Their solution properties in organic solvent were characterized by capillary viscometry. Water-soluble derivatives were compared to other amphiphilic dextrans obtained using a heterogeneous modification in aqueous medium. The effect of modification conditions on substitution pattern was evidenced.

Synthesis and structural characterization of chitosan nanogels.

Colloidal physical gels of pure chitosan were obtained via an ammonia-induced gelation in a reverse phase emulsion. The water weight fraction and the chitosan concentration in the water phase were optimized so as to yield nanogels with controlled particle size and size distribution. The spherical morphology of the nanogels was established by transmission electron microscopy with negative staining. Wide-angle X-ray scattering experiments showed that these gels were partially crystalline. The electrophoretic mobilities of the particles remained positive up to pH 7, above which the particles aggregated due to the charge neutralization. From the investigation on the colloidal stability of these nanogels in various conditions (pH, salt concentration, temperature), an electrosteric stabilization process of the particles was pointed out, related to the conformation of mobile chitosan chains at the gel-liquid interface. Therefore, the structure of the nanogels was deduced as being core-shell type, a gelified core of neutralized chitosan chains surrounded by partially protonated chains.