The Role of Pulmonary Surfactant Phospholipids in Fibrotic Lung Diseases.

Diffuse parenchymal lung diseases (DPLD) or Interstitial lung diseases (ILD) are a heterogeneous group of lung conditions with common characteristics that can progress to fibrosis. Within this group of pneumonias, idiopathic pulmonary fibrosis (IPF) is considered the most common. This disease has no known cause, is devastating and has no cure. Chronic lesion of alveolar type II (ATII) cells represents a key mechanism for the development of IPF. ATII cells are specialized in the biosynthesis and secretion of pulmonary surfactant (PS), a lipid-protein complex that reduces surface tension and minimizes breathing effort. Some differences in PS composition have been reported between patients with idiopathic pulmonary disease and healthy individuals, especially regarding some specific proteins in the PS; however, few reports have been conducted on the lipid components. This review focuses on the mechanisms by which phospholipids (PLs) could be involved in the development of the fibroproliferative response.

Effect of Cholesterol on Biomimetic Membrane Curvature and Coronavirus Fusion Peptide Encapsulation.

Biomimetic cubic phases can be used for protein encapsulation in a variety of applications such as biosensors and drug delivery. Cubic phases with a high concentration of cholesterol and phospholipids were obtained herein. It is shown that the cubic phase structure can be maintained with a higher concentration of biomimetic membrane additives than has been reported previously. Opposing effects on the curvature of the membrane were observed upon the addition of phospholipids and cholesterol. Furthermore, the coronavirus fusion peptide significantly increased the negative curvature of the biomimetic membrane with cholesterol. We show that the viral fusion peptide can undergo structural changes leading to the formation of hydrophobic α-helices that insert into the lipid bilayer. This is of high importance, as a fusion peptide that induces increased negative curvature as shown by the formation of inverse hexagonal phases allows for greater contact area between two membranes, which is required for viral fusion to occur. The cytotoxicity assay showed that the toxicity toward HeLa cells was dramatically decreased when the cholesterol or peptide level in the nanoparticles increased. This suggests that the addition of cholesterol can improve the biocompatibility of the cubic phase nanoparticles, making them safer for use in biomedical applications. As the results, this work improves the potential for the biomedical end-use applications of the nonlamellar lipid nanoparticles and shows the need of systematic formulation studies due to the complex interplay of all components.

1-O-Octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV-2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV-2 Activity.

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.

Phospholipid remodeling and its derivatives are associated with COVID-19 severity.

BACKGROUND: Timely medical intervention in severe cases of coronavirus disease 2019 (COVID-19) and better understanding of the disease's pathogenesis are essential for reducing mortality, but early classification of severe cases and its progression is challenging. OBJECTIVE: We investigated the levels of circulating phospholipid metabolites and their relationship with COVID-19 severity, as well as the potential role of phospholipids in disease progression. METHODS: We performed nontargeted lipidomic analysis of plasma samples (n = 150) collected from COVID-19 patients (n = 46) with 3 levels of disease severity, healthy individuals, and subjects with metabolic disease. RESULTS: Phospholipid metabolism was significantly altered in COVID-19 patients. Results of a panel of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) and of phosphatidylethanolamine and lysophosphatidylethanolamine (LPE) ratios were significantly correlated with COVID-19 severity, in which 16 phospholipid ratios were shown to distinguish between patients with severe disease, mild disease, and healthy controls, 9 of which were at variance with those in subjects with metabolic disease. In particular, relatively lower ratios of circulating (PC16:1/22:6)/LPC 16:1 and (PE18:1/22:6)/LPE 18:1 were the most indicative of severe COVID-19. The elevation of levels of LPC 16:1 and LPE 18:1 contributed to the changes of related lipid ratios. An exploratory functional study of LPC 16:1 and LPE 18:1 demonstrated their ability in causing membrane perturbation, increased intracellular calcium, cytokines, and apoptosis in cellular models. CONCLUSION: Significant Lands cycle remodeling is present in patients with severe COVID-19, suggesting a potential utility of selective phospholipids with functional consequences in evaluating COVID-19's severity and pathogenesis.

Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations.

The efficacies of modern gene-therapies strongly depend on their contents. At the same time the most potent formulations might not contain the best compounds. In this work we investigated the effect of phospholipids and their saturation on the binding ability of (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA) to model membranes at the neutral pH. We discovered that DLin-MC3-DMA has affinity to the most saturated monocomponent lipid bilayer 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an aversion to the unsaturated one 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The preference to a certain membrane was also well-correlated to the phase transition temperatures of phospholipid bilayers, and to their structural and dynamical properties. Additionally, in the case of the presence of DLin-MC3-DMA in the membrane with DOPC the ionizable lipid penetrated it, which indicates possible synergistic effects. Comparisons with other ionizable lipids were performed using a model lipid bilayer of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC). Particularly, the lipids heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102) and [(4-hydroxybutyl) azanediyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) from modern mRNA-vaccines against COVID-19 were investigated and force fields parameters were derived for those new lipids. It was discovered that ALC-0315 binds strongest to the membrane, while DLin-MC3-DMA is not able to reside in the bilayer center. The ability to penetrate the membrane POPC by SM-102 and ALC-0315 can be related to their saturation, comparing to DLin-MC3-DMA.

Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Scramblases and virus infection.

The asymmetric distribution of lipids, maintained by flippases/floppases and scramblases, plays a pivotal role in various physiologic processes. Scramblases are proteins that move phospholipids between the leaflets of the lipid bilayer of the cellular membrane in an energy-independent manner. Recent studies have indicated that viral infection is closely related to cellular lipid distribution. The level and distribution of phosphatidylserine (PtdSer) in cells have been demonstrated to be critical regulators of viral infections. Previous studies have supported that the infection of human immunodeficiency virus (HIV), Zika virus, Ebola virus (EBOV), influenza virus, and dengue fever virus require the externalization of phospholipids mediated by scramblases, which are also involved in the pathogenicity of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we review the relationship of scramblases with viruses and the potential viral effector proteins that might utilize host scramblases.

Lipidomics Revealed Plasma Phospholipid Profile Differences between Deceased and Recovered COVID-19 Patients.

Thorough understanding of metabolic changes, including lipidome alteration, associated with the development of COVID-19 appears to be crucial, as new types of coronaviruses are still reported. In this study, we analyzed the differences in the plasma phospholipid profiles of the deceased COVID-19 patients, those who recovered and healthy people. Due to identified abnormalities in plasma phospholipid profiles, deceased patients were further divided into two subgroups (D1 and D2). Increased levels of phosphatidylethanolamines (PE), phosphatidylcholines (PC) and phosphatidylserines (PS) were found in the plasma of recovered patients and the majority of deceased patients (first subgroup D1) compared to the control group. However, abundances of all relevant PE, PC and PS species decreased dramatically in the plasma of the second subgroup (D2) of five deceased patients. These patients also had significantly decreased plasma COX-2 activity when compared to the control, in contrast to unchanged and increased COX-2 activity in the plasma of the other deceased patients and recovered patients, respectively. Moreover, these five deceased patients were characterized by abnormally low CRP levels and tremendous increase in LDH levels, which may be the result of other pathophysiological disorders, including disorders of the immune system, liver damage and haemolytic anemia. In addition, an observed trend to decrease the autoantibodies against oxidative modifications of low-density lipoprotein (oLAb) titer in all, especially in deceased patients, indicate systemic oxidative stress and altered immune system that may have prognostic value in COVID-19.

Diversified Effects of COVID-19 as a Consequence of the Differential Metabolism of Phospholipids and Lipid Peroxidation Evaluated in the Plasma of Survivors and Deceased Patients upon Admission to the Hospital.

As a result of SARS-CoV-2 infection, inflammation develops, which promotes oxidative stress, leading to modification of phospholipid metabolism. Therefore, the aim of this study is to compare the effects of COVID-19 on the levels of phospholipid and free polyunsaturated fatty acids (PUFAs) and their metabolites produced in response to reactions with reactive oxygen species (ROS) and enzymes (cyclooxygenases-(COXs) and lipoxygenase-(LOX)) in the plasma of patients who either recovered or passed away within a week of hospitalization. In the plasma of COVID-19 patients, especially of the survivors, the actions of ROS and phospholipase A2 (PLA2) cause a decrease in phospholipid fatty acids level and an increase in free fatty acids (especially arachidonic acid) despite increased COXs and LOX activity. This is accompanied by an increased level in lipid peroxidation products (malondialdehyde and 8-isoprostaglandin F2α) and lipid mediators generated by enzymes. There is also an increase in eicosanoids, both pro-inflammatory as follows: thromboxane B2 and prostaglandin E2, and anti-inflammatory as follows: 15-deoxy-Δ-12,14-prostaglandin J2 and 12-hydroxyeicosatetraenoic acid, as well as endocannabinoids (anandamide-(AEA) and 2-arachidonylglycerol-(2-AG)) observed in the plasma of patients who recovered. Moreover, the expression of tumor necrosis factor α and interleukins (IL-6 and IL-10) is increased in patients who recovered. However, in the group of patients who died, elevated levels of N-oleoylethanolamine and N-palmitoylethanolamine are found. Since lipid mediators may have different functions depending on the onset of pathophysiological processes, a stronger pro-inflammatory response in patients who have recovered may be the result of the defensive response to SARS-CoV-2 in survivors associated with specific changes in the phospholipid metabolism, which could also be considered a prognostic factor.

Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes.

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

Pathophysiology of Antiphospholipid Syndrome.

The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, ß2-glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the "lupus anticoagulant." This is not a single entity but rather a family of antibodies directed against complex antigens consisting of ß2-glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.

Direct low field J-edited diffusional proton NMR spectroscopic measurement of COVID-19 inflammatory biomarkers in human serum.

A JEDI NMR pulse experiment incorporating relaxational, diffusional and J-modulation peak editing has been implemented for a low field (80 MHz proton resonance frequency) spectrometer system to measure quantitatively two recently discovered plasma markers of SARS-CoV-2 infection and general inflammation. JEDI spectra capture a unique signature of two biomarker signals from acetylated glycoproteins (Glyc) and the supramolecular phospholipid composite (SPC) signals that are relatively enhanced by the combination of relaxation, diffusion and J-editing properties of the JEDI experiment that strongly attenuate contributions from the other molecular species in plasma. The SPC/Glyc ratio data were essentially identical in the 600 MHz and 80 MHz spectra obtained (R2 = 0.97) and showed significantly different ratios for control (n = 28) versus SARS-CoV-2 positive patients (n = 29) (p = 5.2 × 10-8 and 3.7 × 10-8 respectively). Simplification of the sample preparation allows for data acquisition in a similar time frame to high field machines (∼4 min) and a high-throughput version with 1 min experiment time could be feasible. These data show that these newly discovered inflammatory biomarkers can be measured effectively on low field NMR instruments that do not not require housing in a complex laboratory environment, thus lowering the barrier to clinical translation of this diagnostic technology.

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.

Aggregation-Induced Emission Fluorophore-Incorporated Curcumin-Based Ratiometric Nanoprobe for Hypochlorite Detection in Food Matrices.

The development of efficient, economic, reliable, and accurate monitoring of hypochlorite (ClO-) in food matrices is in great demand for food safety assessment, particularly during its massive use against the COVID-19 epidemic. Here, we prepared an aggregation-induced emission (AIE) fluorophore tetraphenylethylene (TPE)-incorporated curcumin-based hybrid ratiometric fluorescence nanoprobe (Curcumin/TPE@HyNPs) through amphiphilic phospholipid polymer-powered nanoprecipitation, which exhibited a fast, highly sensitive, and selective response to the residual ClO- in real food matrices. Because of the inner filter effect (IFE) from curcumin toward TPE inside the nanoprobe, the bright fluorescence of TPE aggregation at ∼437 nm was effectively quenched, along with an enhanced fluorescence of curcumin at ∼478 nm. Once there was a ClO- residue in food matrices, ClO- triggered the oxidation of o-methoxyphenol inside curcumin and led to the almost complete absorption collapse, thereby terminating curcumin fluorescence at ∼478 nm and the IFE process. Accordingly, the fluorescence of TPE at ∼437 nm was recovered. In this case, a ratiometric fluorescent response of Curcumin/TPE@HyNPs toward the residual ClO- in food matrices (e.g., milk) was proposed with a low detection limit of 0.353 µM and a rapid response time of 140.0 s. Notably, the phospholipid polymer as the protection layer effectively reduced/evaded the nonspecific binding of signal reporters inside the nanoprobe, facilitating it to directly monitor the residual ClO- in real food matrices. This work provided a novel approach to utilize the unconventional AIE luminophors for constructing the efficient and reliable early warning mechanisms toward various food contaminants.

Anti-inflammatory and anti-viral actions of anionic pulmonary surfactant phospholipids.

Pulmonary surfactant is a mixture of lipids and proteins, consisting of 90% phospholipid, and 10% protein by weight, found predominantly in pulmonary alveoli of vertebrate lungs. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), are present within the alveoli at very high concentrations, and exert anti-inflammatory effects by regulating multiple Toll like receptors (TLR2/1, TLR4, and TLR2/6) by antagonizing cognate ligand-dependent activation. POPG also attenuates LPS-induced lung injury in vivo. In addition, these lipids bind directly to RSV and influenza A viruses (IAVs) and block interaction between host cells and virions, and thereby prevent viral replication in vitro. POPG and PI also inhibit RSV and IAV infection in vivo, in mice and ferrets. The lipids markedly inhibit SARS-CoV-2 infection in vitro. These findings suggest that both POPG and PI have strong potential to be applied as both prophylaxis and post-infection treatments for problematic respiratory viral infections.

Exploration of Human Serum Lipoprotein Supramolecular Phospholipids Using Statistical Heterospectroscopy in n-Dimensions (SHY-n): Identification of Potential Cardiovascular Risk Biomarkers Related to SARS-CoV-2 Infection.

SARS-CoV-2 infection causes a significant reduction in lipoprotein-bound serum phospholipids give rise to supramolecular phospholipid composite (SPC) signals observed in diffusion and relaxation edited 1H NMR spectra. To characterize the chemical structural components and compartmental location of SPC and to understand further its possible diagnostic properties, we applied a Statistical HeterospectroscopY in n-dimensions (SHY-n) approach. This involved statistically linking a series of orthogonal measurements made on the same samples, using independent analytical techniques and instruments, to identify the major individual phospholipid components giving rise to the SPC signals. Thus, an integrated model for SARS-CoV-2 positive and control adults is presented that relates three identified diagnostic subregions of the SPC signal envelope (SPC1, SPC2, and SPC3) generated using diffusion and relaxation edited (DIRE) NMR spectroscopy to lipoprotein and lipid measurements obtained by in vitro diagnostic NMR spectroscopy and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The SPC signals were then correlated sequentially with (a) total phospholipids in lipoprotein subfractions; (b) apolipoproteins B100, A1, and A2 in different lipoproteins and subcompartments; and (c) MS-measured total serum phosphatidylcholines present in the NMR detection range (i.e., PCs: 16.0,18.2; 18.0,18.1; 18.2,18.2; 16.0,18.1; 16.0,20.4; 18.0,18.2; 18.1,18.2), lysophosphatidylcholines (LPCs: 16.0 and 18.2), and sphingomyelin (SM 22.1). The SPC3/SPC2 ratio correlated strongly (r = 0.86) with the apolipoprotein B100/A1 ratio, a well-established marker of cardiovascular disease risk that is markedly elevated during acute SARS-CoV-2 infection. These data indicate the considerable potential of using a serum SPC measurement as a metric of cardiovascular risk based on a single NMR experiment. This is of specific interest in relation to understanding the potential for increased cardiovascular risk in COVID-19 patients and risk persistence in post-acute COVID-19 syndrome (PACS).

J-Edited DIffusional Proton Nuclear Magnetic Resonance Spectroscopic Measurement of Glycoprotein and Supramolecular Phospholipid Biomarkers of Inflammation in Human Serum.

Proton nuclear magnetic resonance (NMR) N-acetyl signals (Glyc) from glycoproteins and supramolecular phospholipids composite peak (SPC) from phospholipid quaternary nitrogen methyls in subcompartments of lipoprotein particles) can give important systemic metabolic information, but their absolute quantification is compromised by overlap with interfering resonances from lipoprotein lipids themselves. We present a J-Edited DIffusional (JEDI) proton NMR spectroscopic approach to selectively augment signals from the inflammatory marker peaks Glyc and SPCs in blood serum NMR spectra, which enables direct integration of peaks associated with molecules found in specific compartments. We explore a range of pulse sequences that allow editing based on peak J-modulation, translational diffusion, and T2 relaxation time and validate them for untreated blood serum samples from SARS-CoV-2 infected patients (n = 116) as well as samples from healthy controls and pregnant women with physiological inflammation and hyperlipidemia (n = 631). The data show that JEDI is an improved approach to selectively investigate inflammatory signals in serum and may have widespread diagnostic applicability to disease states associated with systemic inflammation.

Emerging Phospholipid Nanobiomaterials for Biomedical Applications to Lab-on-a-Chip, Drug Delivery, and Cellular Engineering.

The design of advanced nanobiomaterials to improve analytical accuracy and therapeutic efficacy has become an important prerequisite for the development of innovative nanomedicines. Recently, phospholipid nanobiomaterials including 2-methacryloyloxyethyl phosphorylcholine (MPC) have attracted great attention with remarkable characteristics such as resistance to nonspecific protein adsorption and cell adhesion for various biomedical applications. Despite many recent reports, there is a lack of comprehensive review on the phospholipid nanobiomaterials from synthesis to diagnostic and therapeutic applications. Here, we review the synthesis and characterization of phospholipid nanobiomaterials focusing on MPC polymers and highlight their attractive potentials for applications in micro/nanofabricated fluidic devices, biosensors, lab-on-a-chip, drug delivery systems (DDSs), COVID-19 potential usages for early diagnosis and even treatment, and artificial extracellular matrix scaffolds for cellular engineering.

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).

Lipid nanoparticles (LNPs) have been established as an essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver messenger RNA (mRNA) coding for the viral spike protein. Out of the four essential components that comprise LNPs, phospholipids represent an underappreciated opportunity for fundamental and translational study. We investigated this avenue by systematically modulating the identity of the phospholipid in LNPs with the goal of identifying specific moieties that directly enhance or hinder delivery efficacy. Results indicate that phospholipid chemistry can enhance mRNA delivery by increasing membrane fusion and enhancing endosomal escape. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that zwitterionic phospholipids mainly aided liver delivery, whereas negatively charged phospholipids changed the tropism of the LNPs from liver to spleen. These results demonstrate that the choice of phospholipid plays a role intracellularly by enhancing endosomal escape, while also driving organ tropism in vivo. These findings were then applied to Selective Organ Targeting (SORT) LNPs to manipulate and control spleen-specific delivery. Overall, selection of the phospholipid in LNPs provides an important handle to design and optimize LNPs for improved mRNA delivery and more effective therapeutics.