A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations.

Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and control the quality of LNPs. The present study is focused on the development and validation of a high-performance liquid chromatography - diode array detector -evaporative light scattering detector (HPLC-DAD/ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs: ionizable lipids, the neutral co-lipid cholesterol, phospholipids, hydrophilic polymer-lipids for colloidal stability (e.g., a PEGylated lipid). In particular, this study focuses on two innovative synthetic lipids: a switchable cationic lipid (CSL3) which has demonstrated in vitro and in vivo siRNA transfection abilities, and the palmitic acid-grafted-poly(ethyloxazoline)5000 (PolyEtOx), used as an alternative polymer to address allergic reactions attributed to PEGylated lipids. The HPLC separation was achieved on a Poroshell C18 column at 50 °C using a step gradient of a mobile phase composed of water/methanol mixtures with 0.1% (v/v) trifluoroacetic acid (TFA). This method was validated following ICH Q2(R1) & (R2) guidelines in terms of linearity (R² ≥ 0.997), precision (relative standard deviation on peak areas < 5% for intermediate repeatability), accuracy (recoveries between 92.9% and 108.5%), and sensitivity. Indeed, low detection and quantitation limits were determined (between 0.02 and 0.04 µg and between 0.04 and 0.10 µg, respectively). Due to its high selectivity, this method allowed the analysis of lipid degradation products produced through degradation studies in basic, acidic, and oxidative conditions. Moreover, the method was successfully applied to the analysis of several liposome formulations at two key steps of the development process. Consequently, the reported HPLC method offers fast, versatile, selective and quantitative analysis of lipids, essential for development optimization, chemical characterization, and stability testing of LNP formulations.

A Nano-Emulsion Platform Functionalized with a Fully Human scFv-Fc Antibody for Atheroma Targeting: Towards a Theranostic Approach to Atherosclerosis.

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a "theranostic approach" that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe-/- mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.

Electro-responsive hydrogels: macromolecular and supramolecular approaches in the biomedical field.

Hydrogels are soft materials of the utmost importance in the biomedical and healthcare fields. Two approaches can be considered to obtain such biomaterials: the macromolecular one and the supramolecular one. In the first, the chemical gel is based on crosslinking while in the second the physical hydrogel is stabilized thanks to noncovalent interactions. Recently, new trends rely on smart devices able to modify their physico-chemical properties under stimulation. Such stimuli-responsive systems can react to internal (i.e. pH, redox potential, enzyme, etc.) or external (i.e. magnetic field, light, electric field, etc.) triggers leading to smart drug release and drug delivery systems, 3D scaffolds or biosensors. Even if some stimuli-responsive biomaterials are currently widely studied, other ones represent a real challenge. Among them, electro-responsive hydrogels, especially obtained via supramolecular approach, are under-developped leaving room for improvement. Indeed, currently known macromolecular electro-responsive systems are reaching some limitations related to their chemical composition, physicochemical properties, mechanical strength, processing technologies, etc. In contrast, the interest for supramolecular hydrogels has risen for the past few years suggesting that they may provide new solutions as electro-responsive soft materials. In this short review, we give a recent non exhaustive survey on macromolecular and supramolecular approaches for electro-responsive hydrogels in the biomedical field.

Synthesis and Intracellular Uptake of Rhodamine-Nucleolipid Conjugates into a Nanoemulsion Vehicle.

Neurodegenerative diseases represent some of the greatest challenges for both basic science and clinical medicine. Due to their prevalence and the lack of known biochemical-based treatments, these complex pathologies result in an increasing societal cost. Increasing genetic and neuropathological evidence indicates that lysosomal impairment may be a common factor linking these diseases, demanding the development of therapeutic strategies aimed at restoring the lysosomal function. Here, we propose the design and synthesis of a nucleolipid conjugate as a nonviral chemical nanovector to specifically target neuronal cells and intracellular organelles. Herein, thymidine, appropriately substituted to increase its lipophilicity, was used as a model nucleoside and a fluorophore moiety, covalently bound to the nucleoside, allowed the monitoring of nucleolipid internalization in vitro. To improve nucleolipid protection and cellular uptake, these conjugates were formulated in nanoemulsions. In vitro biological assays demonstrated cell uptake- and internalization-associated colocalization with lysosomal markers. Overall, this nucleolipid-nanoemulsion-based formulation represents a promising drug-delivery tool to target the central nervous system, able to deliver drugs to restore the impaired lysosomal function.