MALDI versus DESI mass spectrometry imaging of lipids in atherosclerotic plaque.

Mass spectrometry imaging (MSI) is a powerful tool for detecting lipids in tissue sections, with matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) as its key ionization techniques. In this study, we examine how MALDI compares with state-of-the-art DESI ionization in identifying lipids in heterogeneous samples, specifically atherosclerotic plaques. Carotid plaques (n = 4) from patients undergoing endarterectomy were snap-frozen, stored at -80°C, and then sectioned for MSI analysis and H&E staining. Measurements were conducted using a SYNAPT XS mass spectrometer in positive ion mode, employing MALDI with a 2,5-dihydroxybenzoic acid (DHB) matrix and DESI with a methanol: water (98:2) (v/v) solvent. Our comparison covered spectral profiles, sensitivity, and image quality generated by these two techniques. We found that both MALDI and DESI are highly suitable techniques for detecting a wide range of lipids in atherosclerotic plaque sections. DESI-MSI exhibited higher ion counts for most lipid classes than MALDI-MSI and provided sharper images. MALDI detected larger amounts of ceramide and hexosylceramide species, possibly due to its efficient generation of dehydrated ions. In contrast, DESI showed greater peak intensities of cholesteryl ester and triacylglyceride species than MALDI, consistent with reduced fragmentation. These findings establish the relative merits of DESI and MALDI and demonstrate their complementarity as techniques for lipid research in MSI.

Mechanistic insights into how mixing factors govern polyelectrolyte-surfactant complexation in RNA lipid nanoparticle formulation.

HYPOTHESIS: Lipid nanoparticle self-assembly is a complex process that relies on ion pairing between nucleic acids and hydrophobic cationic lipid counterions for encapsulation. The chemical factors influencing this process, such as formulation composition, have been the focus of recent research. However, the physical factors, particularly the mixing protocol, which directly modulates these chemical factors, have yet to be mechanistically examined using a reproducible mixing platform comparable to the industry standard. We here utilize Flash NanoPrecipitation (FNP), a scalable rapid mixing platform, to isolate and systematically investigate how mixing factors influence this complexation step, first by using a model polyelectrolyte-surfactant system and then generalizing to a typical RNA lipid nanoparticle formulation. EXPERIMENTS: Aqueous polystyrene sulfonate (PSS) and cetrimonium bromide (CTAB) solutions are rapidly homogenized using reproducible FNP mixing and controlled flow rates at different stoichiometric ratios and total solids concentrations to form polyelectrolyte-surfactant complexes (PESCs). Then, key mixing factors such as total flow rate, inlet stream relative volumetric flow rate, and magnitude of flow fluctuation are studied using both this PESC system and an RNA lipid nanoparticle formulation. FINDINGS: Fluctuations in flow as low as ± 5 % of the total flow rate are found to severely compromise PESC formation. This result is replicated in the RNA lipid nanoparticle system, which exhibited significant differences in size (132.7 nm vs. 75.6 nm) and RNA encapsulation efficiency (34.0 % vs. 82.8 %) under fluctuating vs. steady flow. We explain these results in light of the chemical variables isolated and studied; slow or nonuniform mixing generates localized concentration gradients that disrupt the balance between the hydrophobic and electrostatic forces that drive complex formation. These experiments contribute to our understanding of the complexation stage of lipid nanoparticle formation and provide practical insights into the importance of developing controlled mixing protocols in industry.

Exploring the impact of lipid nanoparticles on protein stability and cellular proteostasis.

Lipid nanoparticles (LNPs) have become pivotal in advancing modern medicine, from mRNA-based vaccines to gene editing with CRISPR-Cas9 systems. Though LNPs based therapeutics offer promising drug delivery with satisfactory clinical safety profiles, concerns are raised regarding their potential nanotoxicity. Here, we explore the impacts of LNPs on protein stability in buffer and cellular protein homeostasis (proteostasis) in HepG2 cells. First, we show that LNPs of different polyethylene glycol (PEG) molar ratios to total lipid ratio boost protein aggregation propensity by reducing protein stability in cell lysate and blood plasma. Second, in HepG2 liver cells, these LNPs induce global proteome aggregation, as imaged by a cellular protein aggregation fluorescent dye (AggStain). Such LNPs induced proteome aggregation is accompanied by decrease in cellular micro-environmental polarity as quantified by a solvatochromic protein aggregation sensor (AggRetina). The observed local polarity fluctuations may be caused by the hydrophobic contents of LNPs that promote cellular proteome aggregation. Finally, we exploit RNA sequencing analysis (RNA-Seq) to reveal activation of unfolded protein response (UPR) pathway and other proteostasis genes upon LNPs treatment. Together, these findings highlight that LNPs may induce subtle proteome stress by compromising protein stability and proteostasis even without obvious damage to cell viability.

Lipidic drug delivery systems are responsive to the human microbiome.

In vitro and in vivo tests for therapeutic agents are typically conducted in sterile environments, but many target areas for drug delivery are home to thousands of microbial species. Here, we examine the behaviour of lipidic nanomaterials after exposure to representative strains of four bacterial species found in the gastrointestinal tract and skin. Small angle X-ray scattering measurements show that the nanostructure of monoolein cubic and inverse hexagonal phases are transformed, respectively, into inverse hexagonal and inverse micellar cubic phases upon exposure to a strain of live Staphylococcus aureus often present on skin and mucosa. Further investigation demonstrates that enzymatic hydrolysis and cell membrane lipid transfer are both likely responsible for this effect. The structural responses to S. aureus are rapid and significantly reduce the rate of drug release from monoolein-based nanomaterials. These findings are the first to demonstrate how a key species in the live human microbiome can trigger changes in the structure and drug release properties of lipidic nanomaterials. The effect appears to be strain specific, varies from patient to patient and body region to body region, and is anticipated to affect the bioapplication of monoglyceride-based formulations.

Time-resolved small-angle neutron scattering for characterization of molecular exchange in lipid nanoparticle therapeutics.

HYPOTHESIS: Nano-scale dynamics of self-assembled therapeutics play a large role in their biological function. However, assessment of such dynamics remains absent from conventional pharmaceutical characterization. We hypothesize that time-resolved small-angle neutron scattering (TR-SANS) can reveal their kinetic properties. For lipid nanoparticles (LNP), limited molecular motion is important for avoiding degradation prior to entering cells while, intracellularly, enhanced molecular motion is then vital for effective endosomal escape. We propose TR-SANS for quantifying molecular exchange in LNPs and, therefore, enabling optimization of opposing molecular behaviors of a pharmaceutical in two distinct environments. EXPERIMENTS: We use TR-SANS in combination with traditional SANS and small-angle x-ray scattering (SAXS) to experimentally quantify nano-scale dynamics and provided unprecedented insight to molecular behavior of LNPs. FINDINGS: LNPs have molecular exchange dynamics relevant to storage and delivery which can be captured using TR-SANS. Cholesterol exchanges on the time-scale of hours even at neutral pH. As pH drops below the effective pKa of the ionizable lipid, molecular exchange occurs faster. The results give insight into behavior enabling delivery and provide a quantifiable metric by which to compare formulations. Successful analysis of this multi-component system also expands the opportunities for using TR-SANS to characterize complex therapeutics.

Transitioning the production of lipidic mesophase-based delivery systems from lab-scale to robust industrial manufacturing following a risk-based quality by design approach augmented by artificial intelligence.

Lipidic mesophase drug carriers have demonstrated the capacity to host and effectively deliver a wide range of active pharmaceutical ingredients, yet they have not been as extensively commercialized as other lipid-based products, such as liposomal delivery systems. Indeed, scientists are primarily focused on investigating the physics of these systems, especially in biological environments. Meanwhile, the production methods remain less advanced, and researchers are still uncertain about how the manufacturing process might affect the quality of formulations. Bringing these products to the market will require an industrial translation process. In this scenario, we have developed a robust strategy to produce lipidic mesophase-based drug delivery systems using a dual-syringe setup. We identified four critical process parameters in the newly developed method (dual-syringe method), in comparison to eight in the standard production method (gold standard), and we defined their optimal limits following a Quality by Design approach. The robustness and versatility of the proposed method were assessed experimentally by incorporating drugs with diverse physicochemical properties and augmented by machine learning which, by predicting the drug release from lipidic mesophases, reduces the formulation development time and costs.

The augment effects of magnesium hydride on the lipid lowering effect of atorvastatin: an in vivo and in vitro investigation.

There is strong evidence connecting increased serum lipid levels to cardiovascular disorders, including atherosclerosis. Statins is prescribed as the primary medication to decrease lipid levels. Recent research has demonstrated that hydrogen possesses anti-inflammatory and antioxidant properties by modulating the expression of peroxisome proliferator-activated receptor gamma coactivator-1α, ultimately leading to the preservation of lipid homeostasis. Magnesium hydride (MgH2) is a prolonged stable hydrogen storage medium, which can be utilized to investigate its synergistic lipid-lowering effect with statins and its detailed molecular mechanism, both in vivo and in vitro. To ascertain the safety and efficacy of MgH2, we executed a comprehensive research of its influence on both physiological and pathological metrics. We noted a substantial diminution in lipid levels when MgH2 was integrated with atorvastatin, as attested by oil red staining. Furthermore, we scrutinized the regulatory effect of MgH2 on cytochrome P450 3A, which is a metabolic enzyme of statins, and discovered that it could be reduced by the MgH2. Concluding from our results, we propose that MgH2 inhibits the expression of cytochrome P450 3A in the liver and exerts an auxiliary lipid-lowering effect by increasing the blood concentration of statins. By augmenting our comprehension of MgH2's role in ameliorating lipid metabolism, we aspire to develop more promising therapies in the future.

Influence of lipid and metabolite profiles of mitochondrial fraction on pH and color stability of longissimus lumborum muscle with different ultimate beef pH.

This study aimed to explore the differences in the lipidome and mitochondrial fraction metabolome of Nellore cattle meat in different ranges of ultimate pH (pHu) normal (≤5.79), intermediate (5.80 to 6.19) and high (≥ 6.20) after 3- and 21-d postmortem. Instrumental color, myoglobin redox state, oxygen consumption, and metmyoglobin-reducing activity were measured during storage. A total of 472 lipids and 22 mitochondrial fraction metabolites were identified. Beef with high pHu showed positive regulation of ceramides involved in apoptosis and negative regulation of lipid classes related to membrane permeability and stability. In addition, lower carnitine content was noted in high-pHu beef than in normal-pHu beef. Acylcarnitines, phosphatidylinositol, and IMP showed upregulation in beef with intermediate pHu, indicating changes mainly related to energy, purine and pyruvate metabolism. Aging time impacted on the lipid content and metabolites involved in different metabolic pathways. These results provided new insights into beef's mitochondrial fraction lipid and metabolic profile with different pHu. In addition, beef with intermediate pHu differs from beef with high pHu due to changes in energy metabolism.

Effects of phenolic acid grafted-chitosan hydrocolloids on the aldehyde contents from lipid oxidation in golden pompano (Trachinotus blochii) fillets during pan-frying.

The effects of phenolic acid grafted-chitosan hydrocolloids (CS-g-GA/FA) on aldehyde contents from lipid oxidation in golden pompano fillets during pan-frying was investigated with an established high-performance liquid chromatography-mass spectrum method. Results indicated that pan-frying induced profound lipid oxidation and aldehydes generation with propanal, hexanal, nonanal, trans, trans-2,4-decadienal, and 4-hydroxy-2-nonenal as the abundant species. CS-g-FA and CS-g-GA effectively decreased their contents by 23.74-27.42 %, 61.69-67.42 %, 41.83-53.91 %, 29.91-48.79 %, and 61.57-65.39 % after 3 min. Most aldehyde contents decreased with the extension of pan-frying time due to the volatilization and reaction. In terms of substrate depletion, CS-g-phenolic acids effectively inhibited unsaturated fatty acids oxidation due to their decent antioxidant activity than CS. The significant lower retention rates of aldehydes in the CS-g-phenolic acids groups compared with control in chemical mode confirmed the carbonyl ammonia condensation. These results suggested that CS-g-phenolic acids serve as novel coating to reduce hazardous compounds during aquatic products thermal processing.

Meeting the challenge of varietal and geographical authentication of hazelnuts through lipid metabolite fingerprinting.

Hazelnuts are high-quality products with significant economic importance in many European countries. Their market price depends on their qualitative characteristics, which are driven by cultivar and geographical origin, making hazelnuts susceptible to fraud. This study systematically compared two lipidomic fingerprinting strategies for the simultaneous authentication of hazelnut cultivar and provenance, based on the analysis of the unsaponifiable fraction (UF) and triacylglycerol (TAG) profiles by gas chromatography-mass spectrometry coupled with chemometrics. PLS-DA classification models were developed using a large sample set with high natural variability (n = 309) to discriminate hazelnuts by cultivar and origin. External validation results demonstrated the suitability of the UF fingerprint as a hazelnut authentication tool, both tested models showing a high efficiency (>94 %). The correct classification rate of the TAG fingerprinting method was lower (>80 %), but due to its faster analysis time, it is recommended as a complementary screening tool to UF fingerprinting.

Comprehensive physicochemical indicators analysis and quality evaluation model construction for the post-harvest ripening rapeseeds.

Rapeseed (Brassica napus L.) is the second largest globally cultivated oil crop, but the effects of post-harvested ripening on rapeseed quality is unclear and unpredictable. This study reveals the relationship between post-harvest ripening periods (PHR) and physicochemical quality of different rapeseed cultivars using comprehensive physicochemical indicators analysis. The results indicate that PHR led to a gradual decrease in chlorophyll, carotenoid and moisture content but continually increased oil and total phenol content (TPC). Besides, 295 lipid molecules from 13 lipid subclasses were identified, revealing that the relative content of triacylglycerol (TG) was progressively increased while diacylglycerol (DG) demonstrated a consistent decline throughout the PHR. Correlation analysis, hierarchical cluster analysis (HCA) and principal component analysis (PCA) were employed to construct and verify the comprehensive quality evaluation model for rapeseeds in PHR. This paper develops a comprehensive quality evaluation model for post-harvest ripening rapeseeds and advances the development of agricultural products.

Fabrication and characterization of oleic acid/sesame protein isolate/ poly (vinyl) alcohol core-shell nanofibers: Mitigating lipid oxidation by emulsion electrospinning.

Formulated oil-in-water (O/W) emulsions of oleic acid (OA) using sesame protein isolate (SPI) were processed via emulsion electrospinning with poly (vinyl) alcohol (PVA) to fabricate core-shell nanofibers for lipid oxidation prevention. The emulsion droplet size and viscosity increased as the oil volume fraction rose from 5 % to 30 %. The morphology tests and Fourier transform infrared spectroscopy (FTIR) confirmed the uniformity of nanofibers and OA encapsulation with hydrogen bonding. The thermal stability, mechanical properties, and water contact angle (WCA) of the nanofiber films improved with increased OA content. Encapsulation efficiency was 94.76 % and storage stability was maintained for 7 days in 5 % oil fraction nanofibers. The nanofibers showed lower oxidation and superior oxidative resistance to free OA, with the lowest peroxide value (POV, 2.14 mmol/L) and thiobarbituric acid-reactive substances (TBARS, 36.75 µmol/L). In conclusion, the OA/SPI/PVA (PE) core-shell nanofibers via emulsion electrospinning are efficient for fatty acid encapsulation in functional foods.

Lipidomics analysis reveals the effects of Schizochytrium sp. supplementation on the lipid composition of Tan sheep meat.

Schizochytrium sp. (SZ) can potentially be employed in nutritional strategies for producing high-quality sheep meat. However, the effects of SZ on the lipid composition of sheep meat are insufficiently understood. In this study, the effects of SZ supplementation on the lipid profile of Tan sheep meat were evaluated using non-targeted lipidomic techniques. Lipidomics analysis revealed 383 differential lipids (DLs) between the SZ and control groups, and there were six metabolic pathways associated with lipids, including glycerophospholipid metabolism, glycerolipid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, glycine, serine and threonine metabolism, and arachidonic acid metabolism (P < 0.05). Glycerophospholipid metabolism was the core pathway of DLs; we found that phosphatidylcholine, phosphatidylserine, and lysophosphatidylcholine were the crucial lipid metabolites of this pathway. Dietary supplementation with SZ increased n-3 polyunsaturated fatty acid (PUFA), C22:6n-3, and C20:5n-3 (P < 0.05), while it decreased C18:0, saturated fatty acid (SFA), and SFA/PUFA (P < 0.05). These results indicate that SZ supplementation induces positive alterations in the lipid profile of Tan sheep meat, which is beneficial to meat quality and sheds valuable insights into the future development of functional lipids in sheep meat.

Seasonal variation in non-volatile flavor substances of fresh tea leaves (Camellia sinensis) by integrated lipidomics and metabolomics using UHPLC-Q-Exactive mass spectrometry.

Harvest season exerts great influence on tea quality. Herein, the variations in non-volatile flavor substances in spring and summer fresh tea leaves of four varieties were comprehensively investigated by integrating UHPLC-Q-Exactive based lipidomics and metabolomics. A total of 327 lipids and 99 metabolites were detected, among which, 221 and 58 molecules were significantly differential. The molecular species of phospholipids, glycolipids and acylglycerolipids showed most prominent and structure-dependent seasonal changes, relating to polar head, unsaturation and total acyl length. Particularly, spring tea contained higher amount in aroma precursors of highly unsaturated glycolipids and phosphatidic acids. The contents of umami-enhancing amino acids and phenolic acids, e.g., theanine, theogallin and gallotannins, were increased in spring. Besides, catechins, theaflavins, theasinensins and flavone/flavonol glycosides showed diverse changes. These phytochemical differences covered key aroma precursors, tastants and colorants, and may confer superior flavor of black tea processed using spring leaves, which was verified by sensory evaluation.

Unveiling the protein-lipid interaction mechanism: How the sturgeon lipids diminish the surimi gel properties.

Sturgeon, with 4 times higher lipid content than silver carp (ubiquitously applied for surimi production in China), affects surimi gelling properties. However, how the flesh lipids affect gelling properties remains unclear. This study investigated how flesh lipids impact surimi gelling properties and elucidated the interaction mechanism between lipids and proteins. Results revealed yellow meat contains 7 times higher lipids than white meat. Stronger ionic protein-protein interactions were replaced by weaker hydrophobic forces and hydrogen bonds in protein-lipid interaction. Protein-lipid interaction zones encapsulated lipid particles, changing protein structure from α-helix to ß-sheet structure thereby gel structure becomes flexible and disordered, significantly diminishing surimi gel strength. Docking analysis validated fatty acid mainly binding at Ala577, Ile461, Arg231, Phe165, His665, and His663 of myosin. This study first reported the weakened surimi gelling properties from the perspective of free fatty acids and myosin interactions, offering a theoretical basis for sturgeon surimi production.

"All in one" lipid-polymer nanodelivery system for gene therapy of ischemic diseases.

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS.

Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.

Analysis of the Mammalian Lipidome by DMS Shotgun Lipidomics.

Shotgun Lipidomics is a robust methodology for the characterization of the lipidome of complex biological samples. This assay is among the most quantitative lipidomics methods and is capable of surveying a wide breadth of lipid subclasses, both neutral and polar. The shortfalls of the technique include limitations in lipid species characterization and computationally demanding data analysis requiring isotopic and isobaric overlap correction. Differential Mobility Spectrometry (DMS) has demonstrated its utility in enabling acyl tail characterization within a Shotgun Lipidomics experiment. Here, we present a workflow for DMS Shotgun Lipidomics that measures 1400 possible lipid species. It utilizes the Shotgun Lipidomics Assistant (SLA) application, an open-source application that supervises the data analysis for an expansive Shotgun Lipidomics experiment.

Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.

Recent developments in LC-MS instrumentation and analytical technologies together with bioinformatics tools supporting high-throughput processing of large omics datasets significantly enhanced our capabilities and efficiency of identification and quantification of lipids in diverse biological materials. However, each biological matrix is characterized by its unique lipid composition, thus requiring optimization of analytical and bioinformatics workflows for each studied lipidome. Here, we describe an integrated workflow for deep lipidome profiling, accurate annotation, and semi-absolute quantification of complex lipidomes based on reversed phase chromatography and high resolution mass spectrometry. This chapter provides details on selection of the optimal extraction protocol, acquisition of LC-MS/MS data for accurate annotation of lipid molecular species, and design of lipidome-specific mixtures of internal standards to assist quantitative analysis of complex, native lipidomes.