Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

Self-Assembly of Nucleoside-Derived Low-Molecular-Weight Gelators: A Thermodynamics and Kinetics Study on Different Length Scales.

Biocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleoside-derived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent.

Green reversed-phase HPLC development strategy: Application to artesunate and amodiaquine analysis.

A green analytical chemistry strategy is described to develop a reversed-phase high-performance liquid chromatography method for amodiaquine and artesunate analysis using ethanol-based mobile phases. This method development was particularly challenging due to the basicity of amodiaquine and low UV absorption of artesunate, leading to peak asymmetry and detection issues, respectively. UV detection concern was even more challenging due to the baseline drift observed with ethanol in gradient mode. Several green pH modifiers were selected for their ecofriendly character and their impact on peak shape and detection was investigated. The screening of various stationary phases (19 columns) appeared as a relevant and necessary approach to reach satisfactory peak shape of basic compounds. To support the results of this study, some additional compounds related to artesunate and amodiaquine structures were included. Methods were optimized and validated using total error approach with a mobile phase composed of ethanol and 10 mM formic acid using three different stationary phases from different manufacturers, providing flexibility of the quality control approach. Method greenness was assessed using the National Environmental Methods Index, the Green Analytical Procedure Index, and the Analytical Eco-Scale. Finally, artesunate and amodiaquine were successfully analyzed in fixed dose combination tablets.

Green Analytical Methods of Antimalarial Artemether-Lumefantrine Analysis for Falsification Detection Using a Low-Cost Handled NIR Spectrometer with DD-SIMCA and Drug Quantification by HPLC.

Two green analytical approaches have been developed for the analysis of antimalarial fixed dose tablets of artemether and lumefantrine for quality control. The first approach consisted of investigating the qualitative performance of a low-cost handheld near-infrared spectrometer in combination with the principal component analysis as an exploratory tool to identify trends, similarities, and differences between pharmaceutical samples, before applying the data driven soft independent modeling of class analogy (DD-SIMCA) as a one-class classifier for proper drug falsification detection with 100% of both sensitivity and specificity in the studied cases. Despite its limited spectral range and low resolution, the handheld device allowed detecting falsified drugs with no active pharmaceutical ingredient and identifying specifically a pharmaceutical tablet brand name. The second approach was the quantitative analysis based on the green and fast RP-HPLC technique using ethanol as a green organic solvent and acetic acid as a green pH modifier. The optimal separation was achieved in 7 min using a mobile phase composed of ethanol 96% and 10 mM of acetic acid pH 3.35 (63:37, v/v). The developed method was validated according to the total error approach based on an accuracy profile, was applied to the analysis of tablets, and allowed confirming falsified drugs detected by spectroscopy.

Silver Ions Detection via Nucleolipids Self-Assembly.

A novel hybrid bioinspired amphiphile featuring a cytosine moiety, which self-assembles into liposomes can be used to detect silver ions in aqueous media. The coordination of Ag+ ions by the nucleotide moiety increases membrane rigidity, which enhances the fluorescence of a common reporter, Thioflavin T. Ag+ can be sensed even at trace concentrations (3 ppb) with great specificity over other metals ions. These nucleotide based supramolecular structures can be used to detect silver ions in drinking water, demonstrating the robustness of this approach.

Ceftriaxone Absorption Enhancement for Noninvasive Administration as an Alternative to Injectable Solutions.

Neonatal sepsis is a major cause of infant mortality in developing countries because of delayed injectable treatment, making it urgent to develop noninjectable formulations that can reduce treatment delays in resource-limited settings. Ceftriaxone, available only for injection, needs absorption enhancers to achieve adequate bioavailability via nonparenteral administration. This article presents all available data on the nonparenteral absorption of ceftriaxone in humans and animals, including unpublished work carried out by F. Hoffmann-La Roche (Roche) in the 1980s and new data from preclinical studies with rabbits, and discusses the importance of these data for the development of noninjectable formulations for noninvasive treatment. The combined results indicate that the rectal absorption of ceftriaxone is feasible and likely to lead to a bioavailable formulation that can reduce treatment delays in neonatal sepsis. A bile salt, chenodeoxycholate sodium salt (Na-CDC), used as an absorption enhancer at a 125-mg dose, together with a 500-mg dose of ceftriaxone provided 24% rectal absorption of ceftriaxone and a maximal plasma concentration of 21 µg/ml with good tolerance in human subjects. The rabbit model developed can also be used to screen for the bioavailability of other formulations before assessment in humans.

Chromatographic study of nucleoside-lipids by RP-UHPLC-DAD/CAD.

Today, one of the most popular strategies in drug delivery is the encapsulation of therapeutic agents in supramolecular nanosystems formed from amphiphilic molecules. Synthetic nucleoside-lipids, composed of one nucleoside and lipidic chains, constitute promising new amphiphilic excipients under research in the field of pharmaceutical and biomedical applications. The aim of this work was to study the chromatographic behavior of these nucleoside-lipids in reversed-phase HPLC to establish appropriate chromatographic conditions for their analysis in drug delivery systems. The effect of the stationary phase, the organic solvent, the pH* values, and pH modifier nature of the mobile phase were studied on retention, peak shape, and detection. Good chromatographic performance was achieved on both Syncronis® C18 and Acquity® BEH C18 with mobile phases composed of MeOH/water, 95:5 (v/v) mixtures at apparent pH above 5. Dual detection by diode array detection (DAD) and charged aerosol detection (CAD) was investigated. CAD signal was found to be dependent on the type of pH modifiers added to the mobile phase. In isocratic elution, the same order of magnitude of CAD responses was obtained for the tested nucleoside-lipids. This study led to suitable chromatographic conditions for purity and stability studies of nucleoside-lipids. The purity of the synthetized molecules was established to be superior to 98%. Different stability in organic solvents was noticed depending on nucleoside-lipid structure. This first study will allow quantitative applications to establish loading ratio and encapsulation yield in future drug delivery systems composed of nucleoside-lipid-based assemblies.

Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis.

The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol) with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.

Solid Lipid Nanoparticles for Image-Guided Therapy of Atherosclerosis.

Although the application of nanotechnologies to atherosclerosis remains a young field, novel strategies are needed to address this public health issue. In this context, the magnetic resonance imaging (MRI) approach has been gradually investigated in order to enable image-guided treatments. In this contribution, we report a new approach based on nucleoside-lipids allowing the synthesis of solid lipid nanoparticles (SLN) loaded with iron oxide particles and therapeutic agents. The insertion of nucleoside-lipids allows the formation of stable SLNs loaded with prostacycline (PGI2) able to inhibit platelet aggregation. The new SLNs feature better relaxivity properties in comparison to the clinically used contrast agent Feridex, indicating that SLNs are suitable for image-guided therapy.

Oligonucleotide Solid Nucleolipid Nanoparticles against Antibiotic Resistance of ESBL-Producing Bacteria.

Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to apply this strategy by using a novel nanoformulation consisting of DOTAU, a nucleolipid carrier, in an attempt to simultaneously deliver antibiotic and anti-resistance oligonucleotides. Ceftriaxone, a third-generation cephalosporin, was formulated with DOTAU to form an ion pair, and was then nanoprecipitated. The obtained solid nanocapsules were characterized using FT-IR, XRD, HPLC, TEM and DLS techniques and further functionalized by the anti-resistance ONα sequence. To obtain an optimal anti-resistance activity and encapsulation yield, both the formulation protocol and the concentration of ONα were optimized. As a result, monodispersed negatively charged nanoparticles of CFX-DOTAU-ONα with a molar ratio of 10:24:1 were obtained. The minimum inhibitory concentration of these nanoparticles on the resistant Escherichia coli strain was significantly reduced (by 75%) in comparison with that of non-vectorized ONα. All aforementioned results reveal that our nanoformulation can be considered as an efficient and relevant strategy for oligonucleotide intrabacterial delivery in the fight against antibiotic resistance.

Correction: Nguyen et al. Oligonucleotide Solid Nucleolipid Nanoparticles against Antibiotic Resistance of ESBL-Producing Bacteria. Pharmaceutics 2022, 14, 299.

In the original publication [...].

Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

The initial pharmaceutical development of an artesunate/amodiaquine oral formulation for the treatment of malaria: a public-private partnership.

BACKGROUND: Artemisinin-based combination therapy is currently recommended worldwide for the treatment of uncomplicated malaria. Fixed-dose combinations are preferred as they favour compliance. This paper reports on the initial phases of the pharmaceutical development of an artesunate-amodiaquine (ASAQ) bilayer co-formulation tablet, undertaken following pre-formulation studies by a network of scientists and industrials from institutions of both industrialized and low income countries. METHODS: Pharmaceutical development was performed by a research laboratory at the University Bordeaux Segalen, School of Pharmacy, for feasibility and early stability studies of various drug formulations, further transferred to a company specialized in pharmaceutical development, and then provided to another company for clinical batch manufacturing. The work was conducted by a regional public-private not-for-profit network (TropiVal) within a larger Public Private partnership (the FACT project), set up by WHO/TDR, Médecins Sans Frontières and the Drugs for Neglected Disease initiative (DNDi). RESULTS: The main pharmaceutical goal was to combine in a solid oral form two incompatible active principles while preventing artesunate degradation under tropical conditions. Several options were attempted and failed to provide satisfactory stability results: incorporating artesunate in the external phase of the tablets, adding a pH regulator, alcoholic wet granulation, dry granulation, addition of an hydrophobic agent, tablet manufacturing in controlled conditions. However, long-term stability could be achieved, in experimental batches under GMP conditions, by physical separation of artesunate and amodiaquine in a bilayer co-formulation tablet in alu-alu blisters. Conduction of the workplan was monitored by DNDi. CONCLUSIONS: Collaborations between research and industrial groups greatly accelerated the process of development of the bi-layered ASAQ tablet. Lack of public funding was the main obstacle hampering the development process, and no intellectual property right was claimed. This approach resulted in a rapid technology transfer to the drug company Sanofi-Aventis, finalizing the process of development, registration and WHO pre-qualification of the fixed-dose co-formulation together with DNDi. The bi-layered tablet is made available under the names of Coarsucam® and Artesunate amodiaquine Winthrop®, Sanofi-Aventis. The issue related to the difficulty of public institutions to valorise their participation in such initiative by lack of priority and funding of applied research is discussed.

Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry.

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.

Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology.

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system's components.

An analytical study of lipid-oligonucleotide aggregation properties.

Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.

Development of a green HPLC method for the analysis of artesunate and amodiaquine impurities using Quality by Design.

Greening analytical methods has become of great interest in the field of pharmaceutical analysis to protect both the operators' health and the environment. In this work, an innovative methodology combining Quality-by-Design (QbD) and Green Chemistry principles was followed to develop a single, green and robust RP-HPLC method for the quantitative analysis of impurities of both artesunate and amodiaquine drugs. Ethanol was selected as the best ecofriendly alternative solvent in substitution to the commonly used organic solvents such as acetonitrile and methanol. To achieve method objectives, resolutions between the 10 peaks were chosen as critical method attributes (CMAs) to be optimized through QbD approach. Based on a quality risk assessment, pH, temperature, and gradient slope were then selected as critical method parameters (CMPs) and a three level full factorial design was used to model the CMAs as function of the CMPs. Response surface methodology associated to Monte Carlo simulations allowed to determine the method operable domain region (MODR), i.e., the multidimensional combination of CMPs where CMAs simultaneously satisfied specifications (Rs ≥ 1.5) with a probability at least equal to 95 %. Inside the MODR, the working point was chosen based on green criteria, involving a mobile phase composed of ethanol and 10 mM acetic acid only as pH modifier. The method was successfully validated for all impurities using accuracy profile methodology, which was fully compliant with the ICH Q2(R1) requirements. Finally, the method was applied to the analysis of amodiaquine and artesunate impurities in raw materials and formulations.

Analysis of lipid-oligonucleotide conjugates by cyclodextrin-modified capillary zone electrophoresis.

Lipid-oligonucleotide (LONs) based bioconjugates represent an emerging class of therapeutic agents, allowing the delivery of therapeutic oligonucleotide sequences. The LON development requests accurate and efficient analytical methods. In this contribution, LON analysis methods were developed in cyclodextrin-modified capillary zone electrophoresis (CD-CZE). The LONs selected in this study feature different structures, including i) the oligonucleotide length (from 10 to 20 nucleotides), ii) the inter-nucleotide linkage chemistry (phosphodiester PDE or phosphorothioate PTO), and iii) the lipidic part: single- (LONsc) or double-chain (LONdc) lipids. In CD-CZE, the effect of several parameters on the electrophoretic peaks was investigated (buffer, CD, and capillary temperature). The binding interaction between LON and Me-ß-CD was studied in affinity capillary electrophoresis and revealed a 1:1 LON:CD complex. Non-linear regression and three usual linearization methods (y-reciprocal, x-reciprocal, and double-reciprocal) were used to determine the binding constants (K values of 2.5.104 M-1 and 2.0.104 M-1 for LON PDE and LON PTO, respectively). Quantitative methods with good performances and analysis time lower than 5 min were achieved. Importantly, the developed analysis allows a separation between the i) full-length sequence LONs and their truncated sequences, (n-1), (n-2), and (n-4)-mers and ii) LONsc, LONdc and their corresponding unconjugated oligonucleotides. This work highlights the interest of CD-CZE methods for LON analysis.

Determination of artesunate using reversed-phase HPLC at increased temperature and ELSD detection.

Artesunate (ART) determination can be performed by evaporative light scattering detection with mobile phase composed of CH(3)CN/HCOOH 0.01 M (40:60 v/v; pH 2.85). Evaporative light scattering detection instead of UV detection allowed to improve the sensitivity and the LOD. However, the evaporative light scattering detection response of dihydro-artemisinin appears weaker than for ART, whereas with UV detection the response of ART and dihydroartemisinin seemed similar. Constant analysis time was obtained on using the mobile phase with a flow rate of 0.5 mL/min and column temperature at 60 degrees C instead of 0.7 mL/min at room temperature. This led to less solvent consumption. Moreover, decrease in the flow rate and increase in the column temperature were advantageous for higher sensitivity with both evaporative light scattering detection and UV detection. ART determination in rectal gel and suppositories were compared with these different detection modes and similar results were obtained.