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.

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.

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.

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: 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.

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.

Mechanisms involved during the ultrasonically induced depolymerization of chitosan: characterization and control.

The existence of two mechanisms involved in the ultrasonically induced depolymerization of chitosan is evidenced. The first leads to a rapid scission of polymer chains and a lowering of the polydispersity, and the second is responsible for obtaining short polymer chains and oligomers with a polydispersity increase. A systematic experimental study allowed us to identify and quantify the main parameters influencing the chain scission kinetics. Consequently, using a "master curve" approach, a general law of variation of the molecular weight during the depolymerization is proposed. This law can be used in various experimental conditions to easily control the production of chitosan chains of precise length and low polydispersity or a collection of chito-oligosaccharides (COS). Characterization of the latter by (1)H NMR and MALDI-TOF mass spectrometry shows their high purity and an unchanged primary structure.

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.

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.

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.

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.

Effect of the polymer nature on the structural organization of lipid/polymer particle assemblies.

The nano-organized LipoParticle assemblies, consisting of polymer particles coated with lipid layers, are investigated with the aim of evidencing the impact of the particle chemical nature on their physicochemical behavior. To this end, these colloidal systems are elaborated from anionic submicrometer poly(styrene) (P(St)) or poly(lactic acid) (PLA) particles, and lipid mixtures composed of zwitterionic 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP). As revealed by various experimental techniques, such as quasielastic light scattering, zeta potential measurements, transmission electron microscopy, and 1H NMR spectroscopy, the features of both LipoParticle systems are similar when cationic lipid formulations (DPPC/DPTAP mixtures) are used. This result emphasizes the major role of electrostatic interactions as driving forces in the assembly elaboration process. Conversely, the assemblies prepared only with the zwitterionic DPPC lipid are strongly dependent on the particle chemical nature. The structural characteristics of the assemblies based on PLA particles are not controlled and correspond to aggregates, contrary to P(St) particles. To understand this specific phenomenon, and to consequently improve the final organization of these assemblies which are potentially of great interest in biotechnology and biomedicine, numerous investigations are carried out such as the studies of the impact of the ionic strength and the pH of the preparation media, as well as the presence of ethanol (involved in the PLA particle synthesis) or the mean size of the lipid vesicles. From the resulting data and according to the nature of spherical solid support, hydrophobic effects, hydrogen bonds, or dipole-dipole interactions would also appear to influence the LipoParticle elaboration in the case of zwitterionic lipid formulation.

Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers.

The preparation of a homogeneous series of chitin/chitosan oligomers (chito-oligomers) with the same distribution of degrees of polymerization (DP) ranging from 2 to 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described. This DA-series was obtained according to a two-step chemical process involving (i) the production of a well-defined mixture of glucosamine (GlcN) oligomers obtained by acid hydrolysis of a fully N-deacetylated chitosan and after selective precipitations of the hydrolysis products, and (ii) the partial N-acetylation of the GlcN units of these oligomers from a hydro-alcoholic solution of acetic anhydride in a controlled manner. The characterization of this series of samples with different DAs by proton nuclear magnetic resonance (1H NMR) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed us to determine their average DA and identify the main oligomer structures constituting each mixture. Furthermore, MALDI-TOF MS was particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA for the main DPs from 3 to 7. The modeling of these distributions by means of a binomial law displayed that the chemical N-acetylation of low DP GlcN oligomers, produced in a homogeneous medium, occurs randomly along the oligosaccharide chains in accordance with a statistical (Bernoullian) arrangement. In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP.

An overview of lipid membrane supported by colloidal particles.

In recent years, original hybrid assemblies composed of a particle core surrounded by a lipid shell emerged as promising entities for various biotechnological applications. Their broadened bio-potentialities, ranging from model membrane systems or biomolecule screening supports, to substance delivery reservoirs or therapeutic vectors, are furthered by their versatility of composition due to the possible wide variation in the particle nature and size, as well as in the lipid formulation. The synthesis, the characteristics, and the uses of these Lipid/Particle assemblies encountered in the literature so far are reviewed, and classified according to the spherical core size in order to highlight general trends. Moreover, several criteria are particularly discussed: i) the interactions involved between the particles and the lipids, and implicitly the assembly elaboration mechanism, ii) the most suited techniques for an accurate characterization of the entities from structural and physicochemical points of view, and iii) the remarkable properties of the solid-supported lipid membrane obtained.

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.

Hydrolyzable p(DMAPEMA) polymers for gene delivery.

The efficiency of cationic polymers as transfectants is thought to be closely related to their DNA association/dissociation properties. An incomplete polymer-DNA dissociation could explain the relatively low gene expression obtained with p(DMAEMA) polymers. Our approach was to synthesize a p(DMAEMA) analogue, p(DMAPEMA), bearing an hydrolyzable cationic group incorporated into the pendant chain with a view to improving transfection. The complexation of DNA with both polymers was studied by agarose gel electrophoresis, size and zeta potential measurements, as well as the dissociation of the polyplexes, after treatment by an anionic polymer, sodium hydroxide or heat. The transfection efficiencies of the polyplexes were evaluated with 293T and BHK21 cells in comparison with Exgen 500. P(DMAPEMA) polymers were able to complex DNA and to release it in a free intact form after an alkaline treatment or storage at 37 degrees C. Poly(aspartic acid) was unable to dissociate p(DMAPEMA) based polyplexes, in contrast to p(DMAEMA) ones. No transfection was obtained with p(DMAPEMA) with both cell lines. A slow hydrolysis under physiological conditions resulting in the absence of DNA unpacking or endosomal entrapment could explain these results. Better transfection results were obtained with polyplexes which were able to be dissociated by electrostatic interactions rather than ones which required the hydrolysis mechanism to release free DNA into cells. Scheme of hydrolyzable p(DMAPEMA) polymer.

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.