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The full potential of ionizable lipid nanoparticles (LNPs) as an in vivo nucleic acid delivery platform has not yet been realized given that LNPs primarily accumulate in the liver following systemic administration, limiting their success to liver-centric conditions. The engineering of LNPs with antibody targeting moieties can enable extrahepatic tropism by facilitating site-specific LNP tethering and driving preferential LNP uptake into receptor-expressing cell types via receptor-mediated endocytosis. Obstetric conditions stemming from placental dysfunction, such as preeclampsia, are characterized by overexpression of cellular receptors, including the epidermal growth factor receptor (EGFR), making targeted LNP platforms an exciting potential treatment strategy for placental dysfunction during pregnancy. Herein, an EGFR antibody-conjugated LNP (aEGFR-LNP) platform was developed by engineering LNPs with increasing densities of antibody functionalization. aEGFR-LNPs were screened in vitro in immortalized placental trophoblasts and in vivo in non-pregnant and pregnant mice and compared to non-targeted formulations for extrahepatic, antibody-targeted mRNA LNP delivery to the placenta. Our top performing LNP with an intermediate density of antibody functionalization (1:5 aEGFR-LNP) mediated a ∼twofold increase in mRNA delivery in murine placentas and a ∼twofold increase in LNP uptake in EGFR-expressing trophoblasts compared to non-targeted counterparts. These results demonstrate the potential of antibody-conjugated LNPs for achieving extrahepatic tropism, and the ability of aEGFR-LNPs in promoting mRNA delivery to EGFR-expressing cell types in the placenta. [Display omitted] • Strain-promoted azide-DBCO cycloaddition produces stable antibody-conjugated LNPs • Pregnancy alters EGFR antibody-conjugated LNP biodistribution in vivo • Intermediate EGFR antibody density enhances mRNA LNP delivery to the placenta • EGFR antibody-conjugated LNPs enhance uptake in EGFR-expressing trophoblasts [ABSTRACT FROM AUTHOR]

The stemness loss-associated dysregeneration of impaired alveolar type 2 epithelial (AT2) cells abolishes the reversible therapy of idiopathic pulmonary fibrosis (IPF). We here report an inhalable mucus-penetrating lipid nanoparticle (LNP) for codelivering dual mRNAs, promoting realveolarization via restoring AT2 stemness for IPF treatment. Inhalable LNPs were first formulated with dipalmitoylphosphatidylcholine and our in-house-made ionizable lipids for high-efficiency pulmonary mucus penetration and codelivery of dual messenger RNAs (mRNAs), encoding cytochrome b5 reductase 3 and bone morphogenetic protein 4, respectively. After being inhaled in a bleomycin model, LNPs reverses the mitochondrial dysfunction through ameliorating nicotinamide adenine dinucleotide biosynthesis, which inhibits the accelerated senescence of AT2 cells. Concurrently, pathological epithelial remodeling and fibroblast activation induced by impaired AT2 cells are terminated, ultimately prompting alveolar regeneration. Our data demonstrated that the mRNA-LNP system exhibited high protein expression in lung epithelial cells, which markedly extricated the alveolar collapse and prolonged the survival of fibrosis mice, providing a clinically viable strategy against IPF. [ABSTRACT FROM AUTHOR]

The advent of lipid nanoparticles (LNPs) containing ionizable cationic lipids has enabled the encapsulation, stabilization, and intracellular delivery of nucleic acid payloads, leading to FDA-approved siRNA-based therapy and mRNA-based vaccines. Other nucleic acid-based therapeutic modalities, including protein replacement and CRISPR-mediated gene knockout and editing, are being tested in clinical trials, in many cases, for the treatment of liver-related diseases. However, to fully exploit these therapies beyond the liver, improvements in their delivery to extrahepatic targets are needed. Towards this end, both active targeting strategies based on targeting ligands grafted onto LNPs and passive targeting relying on physicochemical LNP parameters such as surface composition, charge, and size are being evaluated. Often, the latter strategy depends on the interaction of LNPs with blood components, forming what is known as the biomolecular corona. Here, I discuss potential challenges related to current LNP-based targeting strategies and the studies of the biomolecular corona on LNPs. I propose potential solutions to overcome some of these obstacles and present approaches currently being tested in preclinical and clinical studies, which face fewer biological barriers than traditional organ-targeting approaches. [Display omitted] • Presents an overview of LNP-based strategies for extrahepatic RNA delivery. • Highlights potential challenges with the LNP strategies and approaches to overcome some of them. • Stresses inherent challenges associated with biomolecular corona studies of LNPs. • Highlights that LNPs are a minor subpopulation of nanoparticles in human plasma. • Advocates innovative approaches to drive progress in the LNP field. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) has emerged as a promising therapeutic modality for various diseases. However, efficient delivery of mRNA into target cells remains a significant challenge. In this study, the combinatorial synthesis and characterization of a novel series of ionizable biscarbamate lipids (IBLs) for mRNA lipid nanoparticle (LNP) delivery are reported. A simplified and scalable method is developed, resulting in IBLs suitable for formulating mRNA into stable LNPs. Two generations of IBLs are synthesized and evaluated for their mRNA transfection capacity in vitro, using eGFP as a reporter protein, leading to the identification of S‐Ac7‐DOG as a lead IBL. Upon intramuscular vaccination, S‐Ac7‐DOG LNPs instigated robust antigen‐specific CD8+ T cell responses against an mRNA encoded viral oncoprotein and a tumor neo‐antigen. In comparison to MC3 LNPs, which are used as a benchmark, S‐Ac7‐DOG LNPs exhibit low reactogenicity, robust mRNA transfection, and a distinct biodistribution, with higher accumulation in draining lymph nodes and spleen. These findings highlight the potential of IBLs as a novel and promising class of ionizable lipids for mRNA delivery in vaccines and beyond. [ABSTRACT FROM AUTHOR]

Despite the widespread use of ionizable lipid nanoparticles (LNPs) in clinical applications for messenger RNA (mRNA) delivery, the mRNA drug delivery system faces an efficient challenge in the screening of LNPs. Traditional screening methods often require a substantial amount of experimental time and incur high research and development costs. To accelerate the early development stage of LNPs, we propose TransLNP, a transformer-based transfection prediction model designed to aid in the selection of LNPs for mRNA drug delivery systems. TransLNP uses two types of molecular information to perceive the relationship between structure and transfection efficiency: coarse-grained atomic sequence information and fine-grained atomic spatial relationship information. Due to the scarcity of existing LNPs experimental data, we find that pretraining the molecular model is crucial for better understanding the task of predicting LNPs properties, which is achieved through reconstructing atomic 3D coordinates and masking atom predictions. In addition, the issue of data imbalance is particularly prominent in the real-world exploration of LNPs. We introduce the BalMol block to solve this problem by smoothing the distribution of labels and molecular features. Our approach outperforms state-of-the-art works in transfection property prediction under both random and scaffold data splitting. Additionally, we establish a relationship between molecular structural similarity and transfection differences, selecting 4267 pairs of molecular transfection cliffs, which are pairs of molecules that exhibit high structural similarity but significant differences in transfection efficiency, thereby revealing the primary source of prediction errors. The code, model and data are made publicly available at https://github.com/wklix/TransLNP. [ABSTRACT FROM AUTHOR]

Despite research efforts being made towards preserving (or even regenerating) heart tissue after an ischemic event, there is a lack of resources in current clinical treatment modalities for patients with acute myocardial infarction that specifically address cardiac tissue impairment. Modified messenger RNA (modRNA) presents compelling properties that could allow new therapeutic strategies to tackle the underlying molecular pathways that ultimately lead to development of chronic heart failure. However, clinical application of modRNA for the heart is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of RNA delivery systems, which were recently approved for clinical usage in mRNA-based COVID-19 vaccines. In this study, we evaluated the potential of LNPs for cardiac delivery of modRNA. We tested how variations in C12–200 modRNA-LNP composition affect transfection levels and biodistribution after intramyocardial administration in both healthy and myocardial-infarcted mice, and determined the targeted cardiac cell types. Our data revealed that LNP-mediated modRNA delivery outperforms the current state of the art (modRNA in citrate buffer) upon intramyocardial administration in mice, with only minor differences among the formulations tested. Furthermore, we determined both in vitro and in vivo that the cardiac cells targeted by modRNA-LNPs include fibroblasts, endothelial cells and epicardial cells, suggesting that these cell types could represent targets for therapeutic interference with these LNP formulations. These outcomes may serve as a starting point for LNP development specifically for therapeutic mRNA cardiac delivery applications. [Display omitted] [ABSTRACT FROM AUTHOR]

BACKGROUND: Exposure to obesogenic chemicals has been reported to result in enhanced adipogenesis, higher adipose tissue accumulation, and reduced ovarian hormonal synthesis and follicular function. We have reported that organotins [tributyltin (TBT) and triphenyltin (TPT)] dysregulate cholesterol trafficking in ovarian theca cells, but, whether organotins also exert lipogenic effects on ovarian cells remains unexplored. OBJECTIVE: We investigated if environmentally relevant exposures to organotins [TBT, TPT, or dibutyltin (DBT)] induce lipid dysregulation in ovarian theca cells and the role of the liver X receptor (LXR) in this effect. We also tested the effect of TBT on oocyte maturation and neutral lipid accumulation, and lipid-related transcript expression in cumulus cells and preimplantation embryos. METHODS: Primary theca cell cultures derived from human and ovine ovaries were exposed to TBT, TPT, or DBT (1, 10, or 50 ng/ml). The effect of these chemical exposures on neutral lipid accumulation, lipid abundance and composition, lipid homeostasis-related gene expression, and cytokine secretion was evaluated using liquid chromatography–mass spectrometry (LC-MS), inhibitor-based methods, cytokine secretion, and lipid ontology analyses. We also exposed murine cumulus-oocyte complexes to TBT and evaluated oocyte maturation, embryo development, and lipid homeostasis-related mRNA expression in cumulus cells and blastocysts. RESULTS: Exposure to TBT resulted in higher intracellular neutral lipids in human and ovine primary theca cells. In ovine theca cells, this effect was dose-dependent, independent of cell stage, and partially mediated by LXR. DBT and TPT resulted in higher intracellular neutral lipids but to a lesser extent in comparison with TBT. More than 140 lipids and 9 cytokines were dysregulated in TBT-exposed human theca cells. Expression of genes involved in lipogenesis and fatty acid synthesis were higher in theca cells, as well as in cumulus cells and blastocysts exposed to TBT. However, TBT did not impact the rates of oocyte maturation or blastocyst development. DISCUSSION: TBT induced dyslipidemia in primary human and ovine theca cells, which may be responsible for some of the TBT-induced fertility dysregulations reported in rodent models of TBT exposure. [ABSTRACT FROM AUTHOR]

Insufficient accumulation of lipid nanoparticles (LNPs)‐based mRNA vaccines in antigen presenting cells remains a key barrier to eliciting potent antitumor immune responses. Herein, we develop dendritic cells (DCs) targeting LNPs by taking advantage of mannose receptor‐mediated endocytosis. Efficient delivery of mRNA to DCs is achieved in vitro and in vivo utilizing the sweet LNPs (STLNPs‐Man). Intramuscular injection of mRNA vaccine (STLNPs‐Man@mRNAOVA) results in a four‐fold higher uptake by DCs in comparison with commercially used LNPs. Benefiting from its DCs targeting ability, STLNPs‐Man@mRNAOVA significantly promotes the antitumor performances, showing a comparable therapeutic efficacy by using one‐fifth of the injection dosage as the vaccine prepared from normal LNPs, thus remarkably avoiding the side effects brought by conventional mRNA vaccines. More intriguingly, STLNPs‐Man@mRNAOVA exhibits the ability to downregulate the expression of cytotoxic T‐lymphocyte‐associated protein 4 on T cells due to the blockade of CD206/CD45 axis, showing brilliant potentials in promoting antitumor efficacy combined with immune checkpoint blockade therapy. [ABSTRACT FROM AUTHOR]

Insufficient accumulation of lipid nanoparticles (LNPs)‐based mRNA vaccines in antigen presenting cells remains a key barrier to eliciting potent antitumor immune responses. Herein, we develop dendritic cells (DCs) targeting LNPs by taking advantage of mannose receptor‐mediated endocytosis. Efficient delivery of mRNA to DCs is achieved in vitro and in vivo utilizing the sweet LNPs (STLNPs‐Man). Intramuscular injection of mRNA vaccine (STLNPs‐Man@mRNAOVA) results in a four‐fold higher uptake by DCs in comparison with commercially used LNPs. Benefiting from its DCs targeting ability, STLNPs‐Man@mRNAOVA significantly promotes the antitumor performances, showing a comparable therapeutic efficacy by using one‐fifth of the injection dosage as the vaccine prepared from normal LNPs, thus remarkably avoiding the side effects brought by conventional mRNA vaccines. More intriguingly, STLNPs‐Man@mRNAOVA exhibits the ability to downregulate the expression of cytotoxic T‐lymphocyte‐associated protein 4 on T cells due to the blockade of CD206/CD45 axis, showing brilliant potentials in promoting antitumor efficacy combined with immune checkpoint blockade therapy. [ABSTRACT FROM AUTHOR]

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1β-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) largely rely on ionizable lipids to yield successful nucleic acid delivery via electrostatic disruption of the endosomal membrane. Here, we report the identification and evaluation of ionizable lipids containing a thiophene moiety (Thio-lipids). The Thio-lipids can be readily synthesized via the Gewald reaction, allowing for modular lipid design with functional constituents at various positions of the thiophene ring. Through the rational design of ionizable lipid structure, we prepared 47 Thio-lipids and identified some structural criteria required in Thio-lipids for efficient mRNA (messenger RNA) encapsulation and delivery in vitro and in vivo. Notably, none of the tested lipids have a pH-response profile like traditional ionizable lipids, potentially due to the electron delocalization in the thiophene core. Placement of the tails and localization of the ionizable headgroup in the thiophene core can endow the nanoparticles with the capability to reach various tissues. Using high-throughput formulation and barcoding techniques, we optimized the formulations to select two top lipids--20b and 29d--and investigated their biodistribution in mice. Lipid 20b enabled LNPs to transfect the liver and spleen, and 29d LNP transfected the lung and spleen. Unexpectedly, LNP with lipid 20b was especially potent in mRNA delivery to the retina with no acute toxicity, leading to the successful delivery to the photoreceptors and retinal pigment epithelium in non-human primates. [ABSTRACT FROM AUTHOR]

Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs). Methods: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure. Results: Cryo-TEM and DLS complemented SAXS, revealed a core–shell mRNA-LNP structure with electron-rich mRNA-rich region at the core, surrounded by lipids. The reconstructed model, utilizing the DENSS algorithm, effectively distinguishes mRNA and lipids via electron density mapping. Notably, DENSS accurately models the morphology of the mRNA-LNPs as an ellipsoidal shape with a "bleb" architecture or a two-compartment structure with contrasting electron densities, corresponding to mRNA-filled and empty lipid compartments, respectively. Finally, subtle changes in the LNP structure after three freeze–thaw cycles were detected by SAXS, demonstrating an increase in radius of gyration (Rg) associated with mRNA leakage. Conclusion: Analyzing SAXS profiles based on DENSS algorithm to yield a reconstructed electron density based three-dimensional model can be a useful physicochemical characterization method in the toolbox to study mRNA-LNPs and facilitate their development. [ABSTRACT FROM AUTHOR]

mRNA vaccines have emerged as a pivotal tool in combating COVID-19, offering an advanced approach to immunization. A key challenge with these vaccines is their need for extremely-low-temperature storage, which affects their stability and shelf life. Our research addresses this issue by enhancing the stability of mRNA vaccines through a novel cationic lipid, O,O′-dimyristyl-N-lysyl aspartate (DMKD). DMKD effectively binds with mRNA, improving vaccine stability. We also integrated phosphatidylserine (PS) into the formulation to boost immune response by promoting the uptake of these nanoparticles by immune cells. Our findings reveal that DMKD-PS nanoparticles maintain structural integrity under long-term refrigeration and effectively protect mRNA. When tested, these nanoparticles containing green fluorescent protein (GFP) mRNA outperformed other commercial lipid nanoparticles in protein expression, both in immune cells (RAW 264.7 mouse macrophage) and non-immune cells (CT26 mouse colorectal carcinoma cells). Importantly, in vivo studies show that DMKD-PS nanoparticles are safely eliminated from the body within 48 h. The results suggest that DMKD-PS nanoparticles present a promising alternative for mRNA vaccine delivery, enhancing both the stability and effectiveness of these vaccines. [ABSTRACT FROM AUTHOR]

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo—the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson–Boltzmann theory to capture these phenomena. [ABSTRACT FROM AUTHOR]

Background: The Zhizi Chuanxiong herb pair (ZCHP) can delay pathological progression of atherosclerosis (AS); however, its pharmacological mechanism remains unclear because of its complex components. The purpose of current study is to systematically investigate the anti-AS mechanism of ZCHP. Methods: The databases of TCMSP, STITCH, SwissTargetPrediction, BATMAN-TCM, and ETCM were searched to predict the potential targets of ZCHP components. Disease targets associated with AS was retrieved from the GEO database. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were executed using DAVID 6.8. Molecular docking method was employed to evaluate the core target binding to blood components, and animal experiments were performed to test action mechanism. Results: A ZCHP-components-targets-AS network was constructed by using Cytoscape, included 11 main components and 52 candidate targets. Crucial genes were shown in the protein–protein interaction network, including TNF, IL-1β, IGF1, MMP9, COL1A1, CCR5, HMOX1, PTGS1, SELE, and SYK. KEGG enrichment illustrated that the NF-κB, Fc epsilon RI, and TNF signaling pathways were important for AS treatment. These results were validated by molecular docking. In ApoE−/− mice, ZCHP significantly reduced intima-media thickness, pulse wave velocity, plaque area, and serum lipid levels while increasing the difference between the end-diastolic and end-systolic diameters. Furthermore, ZCHP significantly decreased the mRNA and protein levels of TNF-α and IL-1β, suppressed NF-κB activation, and inhibited the M1 macrophage polarization marker CD86 in ApoE−/− mice. Conclusion: This study combining network pharmacology, molecular biology, and animal experiments showed that ZCHP can alleviate AS by suppressing the TNF/NF-κB axis and M1 macrophage polarization. [ABSTRACT FROM AUTHOR]

Ionizable lipid nanoparticles (LNPs) pivotal to the success of COVID-19 mRNA (messenger RNA) vaccines hold substantial promise for expanding the landscape of mRNA-based therapies. Nevertheless, the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity. The intricate nature of biological systems and inadequate knowledge of lipid structure-activity relationships emphasize the significance of high-throughput methods to produce chemically diverse lipid libraries for mRNA delivery screening. Here, we introduce a streamlined approach for the rapid design and synthesis of combinatorial libraries of biodegradable ionizable lipids. This led to the identification of iso-A11B5C1, an ionizable lipid uniquely apt for muscle-specific mRNA delivery. It manifested high transfection efficiencies in muscle tissues, while significantly diminishing off-targeting in organs like the liver and spleen. Moreover, iso-A11B5C1 also exhibited reduced mRNA transfection potency in lymph nodes and antigen-presenting cells, prompting investigation into the influence of direct immune cell transfection via LNPs on mRNA vaccine effectiveness. In comparison with SM-102, while iso-A11B5C1' s limited immune transfection attenuated its ability to elicit humoral immunity, it remained highly effective in triggering cellular immune responses after intramuscular administration, which is further corroborated by its strong therapeutic performance as cancer vaccine in a melanoma model. Collectively, our study not only enriches the high-throughput toolkit for generating tissue-specific ionizable lipids but also encourages a reassessment of prevailing paradigms in mRNA vaccine design. This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) have been used as a carrier for messenger RNA (mRNA) vaccines. Surface properties of LNPs are important to the stability and function of mRNA vaccines. Polyethylene-glycol (PEG) is a functional lipid at the surface of LNPs that improves colloidal stability, increases circulation time, and impacts cellular uptake. In this study, we explore in-depth lipid composition at the surface of mRNA-LNPs using high-field nuclear magnetic resonance (NMR) spectroscopy. Our results provide a unique surface lipid profile of intact LNPs identifying PEG chains and partial ionizable lipids are present with quantification capability. The surface PEG density is determined to reveal the brush-like conformation on the surface of mRNA-LNPs. Furthermore, we implement a diffusion NMR strategy for routine testing of formulated drug products during drug development. Comparative NMR analysis of different vaccine preparations and stability samples provides a global view of the mRNA-LNP surface structure for enhanced product knowledge. [ABSTRACT FROM AUTHOR]

Obesity presents a major risk factor in the development of cardiovascular diseases. Recent reports indicate that many kinds of polyphenols have the potential to prevent metabolic diseases. We hypothesized that rose polyphenols (ROSE) have the effect of improvement in lipid metabolism. In this study, we investigated whether rose polyphenols affected lipid metabolism and exerted antiobesity. To clarify the mechanism, C57BL/6J mice were fed a high-fat diet containing 0.25% ROSE for 35 days. Compared with the control group, body weight gain and adipose tissue weight in the 0.25% ROSE group were significantly decreased. Serum cholesterol and hepatic triglyceride concentrations significantly decreased, whereas fecal triglyceride was significantly increased in the 0.25% ROSE group. Liver stearoyl-CoA desaturase 1 (Scd1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and acyl-CoA:cholesterol acyltransferase 1 (Acat1) mRNA as well as protein stearoyl-CoA desaturase 1 concentrations were significantly lower in the 0.25% ROSE group than that in the control group. The mRNA and the protein concentrations of adipose triglyceride lipase, hormone-sensitive lipase, and peroxisomal acylcoenzyme A oxidase 1 in white adipose tissue were significantly higher in the 0.25% ROSE group than that in the control group. The components in rose polyphenols were quantified by liquid chromatography-tandem mass spectrometry, and we consider that ellagic acid plays an important role in an antiobesity effect because the ellagic acid content is the highest among polyphenols in rose polyphenols. In summary, rose polyphenols exhibit antiobesity effects by influencing lipid metabolism-related genes and proteins to promote lipolysis and suppress lipid synthesis. In this study, intake of 0.25% ROSE CRYSTA-70 was beneficial to prevent obesity. A decrease in hepatic TG synthesis was related with the decrease of stearoyl-CoA desaturase 1. Serum cholesterol was decreased, accompanying a decrease of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Total adipose tissue weight was reduced by the activation of adipose TG lipase and hormone-sensitive lipase. Increased excretion of fecal TG was due to the inhibition of TG absorption. Abbreviations: ATGL, adipose triglyceride lipase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HSL, hormone-sensitive lipase; SCD-1, stearoyl-CoA desaturase 1; TG, triglyceride. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] mRNA is a versatile drug molecule with therapeutic applications ranging from protein replacement therapies to in vivo gene engineering. mRNA delivery is often accomplished using lipid nanoparticles, which are formulated via mixing of aqueous and organic solutions. Although this has historically been accomplished by manual mixing for bench scale science, microfluidic mixing is required for scalable continuous manufacturing and batch to batch control. Currently, there is limited understanding on how the mixing process affects mRNA delivery efficacy, particularly in regard to tropism. To address this knowledge gap, we examined the influence of the type of mixing and microfluidic mixing parameters on the performance of lipid nanoparticles in mice. This was accomplished with a Design of Experiment approach using four nanoparticle formulations with varied ionizable lipid chemistry. We found that each formulation required unique optimization of mixing parameters, with the total delivery efficacy of each lipid nanoparticle generated with microfluidics ranging from 100-fold less to 4-fold more than manually mixed LNPs. Further, mixing parameters influenced organ tropism, with the most efficacious formulations disproportionately increasing liver delivery compared to other organs. These data suggest that mixing parameters for lipid nanoparticle production may require optimization for each unique chemical formulation, complicating translational efforts. Further, microfluidic parameters must be chosen carefully to balance overall mRNA delivery efficacy with application-specific tropism requirements. [ABSTRACT FROM AUTHOR]

RNA therapies have recently taken a giant leap forward with the approval of Onpattro™, a siRNA therapy delivered using a lipid nanoparticle (LNP), and the LNP-enabled mRNA vaccines against COVID-19, which are the first mRNA drugs to reach the marketplace. The latter medicines have illustrated that stability is a significant challenge in the distribution of RNA drugs using non-viral delivery systems, particularly in areas without cold chain storage. Here, we describe a proof-of-concept study on the engineering of an LNP mRNA formulation suitable for spray drying. This process produced a dry powder formulation that maintained stability and preserved mRNA functionality with increased performance compared to liquid formulations stored two weeks at 4 °C. Intratracheal delivery of spray dried LNPs loaded with eGFP mRNA to rats resulted in the production of the eGFP protein in a range of cell types including bronchiolar epithelial cells, macrophages and type II pneumocytes; cell types involved in adaptive immunity and which would be valuable targets for inhaled vaccines against respiratory pathogens. Together, these data show that spray drying of LNPs enhances their stability and may enable RNA delivery to the lung for protein replacement therapy, gene editing, vaccination, and beyond. [Display omitted] • Engineering of an LNP formulation suitable for spray drying. • Spray drying of LNPs enhances their stability and enable RNA delivery to the lung • Protein expression detected in cell types involved in adaptive immunity. [ABSTRACT FROM AUTHOR]

mRNA‐based protein replacement therapy has received much attention as a novel intervention in clinical disease treatment. Lipid nanoparticles (LNPs) are widely used for their therapeutic potential to efficiently deliver mRNA. However, clinical translation has been hampered by the immunogenicity of LNPs that may aggravate underlying disease states. Here, we report a novel ionizable LNP with enhanced potency and safety. The piperazine‐based biodegradable ionizable lipid (244cis) was developed for LNP formulation and its level of protein expression and immunogenicity in the target tissue was evaluated. It was found that 244cis LNP enabled substantial expression of the target protein (human erythropoietin), while it minimally induced the secretion of monocyte chemoattractant protein 1 (MCP‐1) as compared to other conventional LNPs. Selective lung targeting of 244cis LNP was further investigated in tdTomato transgenic mice with bleomycin‐induced pulmonary fibrosis (PF). The repeated administration of 244cis LNP with Cre recombinase mRNA achieved complete transfection of lung endothelial cells (~80%) and over 40% transfection of Sca‐1‐positive fibroblasts. It was shown that 244cis LNP allows the repeated dose of mRNA without the loss of activity due to its low immunogenicity. Our results demonstrate that 244cis LNP has great potential for the treatment of chronic diseases in the lungs with improved potency and safety. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) have been used as a carrier for messenger RNA (mRNA) vaccines. Surface properties of LNPs are important to the stability and function of mRNA vaccines. Polyethylene-glycol (PEG) is a functional lipid at the surface of LNPs that improves colloidal stability, increases circulation time, and impacts cellular uptake. In this study, we explore in-depth lipid composition at the surface of mRNA-LNPs using high-field nuclear magnetic resonance (NMR) spectroscopy. Our results provide a unique surface lipid profile of intact LNPs identifying PEG chains and partial ionizable lipids are present with quantification capability. The surface PEG density is determined to reveal the brush-like conformation on the surface of mRNA-LNPs. Furthermore, we implement a diffusion NMR strategy for routine testing of formulated drug products during drug development. Comparative NMR analysis of different vaccine preparations and stability samples provides a global view of the mRNA-LNP surface structure for enhanced product knowledge. [ABSTRACT FROM AUTHOR]

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes. [ABSTRACT FROM AUTHOR]

Multiple sclerosis is a disease caused by autoantigen-responsive immune cells that disrupt the myelin in the central nervous system (CNS). Although immunosuppressive drugs are used to suppress symptoms, no definitive therapy exists. As in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis, a partial sequence of the myelin oligodendrocyte glycoprotein (MOG35–55) was identified as a causative autoantigen. This suggests that the induction of immune tolerance that is specific to MOG35–55 would be a fundamental treatment for EAE. We previously reported that lipid nanoparticles (LNPs) containing an anionic phospholipid, phosphatidylserine (PS), in their lipid composition, can be used to deliver mRNA and that this leads to proteins of interest to be expressed in the spleen. In addition to the targeting capability of PS, PS molecules avoid activating the immune system. Physiologically, the recognition of PS on apoptotic cells suppresses immune activation against these cells by releasing cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β that negatively regulate immunity. In this study, we tested whether mRNA delivery of autoantigens to the spleen by PS-LNPs causes the expression of MOG35–55 antigens with minimal immune stimulation and whether this could be used to treat an EAE model by inducing immune tolerance. [ABSTRACT FROM AUTHOR]

Purpose: Bacillus coagulans GBI-30, 6086 (BC30) was previously shown to improve nutrient digestibility and amino acid absorption from milk protein in vitro. However, the effect of supplementation with this probiotic on lactose digestibility has not yet been evaluated in vivo. Methods: Wistar female rats were exposed to an acute high-lactose diet (LD; 35% lactose) meal challenge after 7 days of administration of BC30 (LD-BC; n = 10) or vehicle (LD-C; n = 10). Rats treated with vehicle and exposed to control diet (CD; 35% corn starch) meal were used as controls (CD-C; n = 10). Carbohydrate oxidation (CH_OX) and lipid oxidation (L_OX) were monitored by indirect calorimetry before and after lactose challenge. After the challenge, rats were treated daily with vehicle or probiotic for an additional week and were fed with CD or LD ad libitum to determine the effects of BC30 administration in a lactose-induced diarrhoea and malnutrition model. Results: LD-C rats showed lower CH_OX levels than CD rats, while LD-BC rats showed similar CH_OX levels compared to CD rats during the lactose challenge, suggesting a better digestion of lactose in the rats supplemented with BC30. BC30 completely reversed the increase in the small intestine length of LD-C animals. LD-BC rats displayed increased intestinal mRNA Muc2 expression. No significant changes were observed due to BC30 administration in other parameters, such as serum calprotectin, intestinal MPO activity, intestinal A1AT and SGLT1 levels or intestinal mRNA levels of Claudin2 and Occludin. Conclusion: Treatment with BC30 improved the digestibility of lactose in an acute lactose challenge and ameliorated some of the parameters associated with lactose-induced malnutrition. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies. [Display omitted] • Lipid nanoparticles (LNPs) achieve ∼100% of mRNA transfected and viable NK cells. • LNPs-mRNA delivery outperforms electroporation and polymeric nanoparticles. • β-sitosterol-LNPs show improved transfection efficiency and expression intensity. • LNPs achieve ∼100% of mRNA transfected and viable T cells. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) can treat genetic disease using protein replacement or genome editing approaches but requires a suitable carrier to circumnavigate biological barriers and access the desired cell type within the target organ. Lipid nanoparticles (LNPs) are widely used in the clinic for mRNA delivery yet are limited in their applications due to significant hepatic accumulation because of the formation of a protein corona enriched in apolipoprotein E (ApoE). Our lab developed selective organ targeting (SORT) LNPs that incorporate a supplementary component, termed a SORT molecule, for tissue-specific mRNA delivery to the liver, spleen, and lungs of mice. Mechanistic work revealed that the biophysical class of SORT molecule added to the LNP forms a distinct protein corona that helps determine where in the body mRNA is delivered. To better understand which plasma proteins could drive tissue-specific mRNA delivery, we characterized a panel of quaternary ammonium lipids as SORT molecules to assess how chemical structure affects the organ-targeting outcomes and protein corona of lung-targeting SORT LNPs. We discovered that variations in the chemical structure of both the lipid alkyl tail and headgroup impact the potency and specificity of mRNA delivery to the lungs. Furthermore, changes to the chemical structure alter the quantities and identities of protein corona constituents in a manner that correlates with organ-targeting outcomes, with certain proteins appearing to promote lung targeting whereas others reduce delivery to off-target organs. These findings unveil a nuanced relationship between LNP chemistry and endogenous targeting, where the ensemble of proteins associated with an LNP can play various roles in determining the tissue-specificity of mRNA delivery, providing further design criteria for optimization of clinically-relevant nanoparticles for extrahepatic delivery of genetic payloads. [Display omitted] [ABSTRACT FROM AUTHOR]

• DS-5670a, an mRNA vaccine, targets the RBD of the SARS-CoV-2 spike protein. • Immunologically-naïve adults received 2 injections of DS-5670a, 4 weeks apart. • Safety, immunogenicity and pharmacokinetics were evaluated in two dose groups. • DS-5670a was well tolerated and most TEAEs were mild or moderate. • 60 µg of mRNA provided greater humoral immunity and will be investigated further. DS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2. The study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated. Most solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10 %) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses. The novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster. Trial registry: https://jrct.niph.go.jp/latest-detail/jRCT2071210086. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1x, 10x, or 256x LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions. [ABSTRACT FROM AUTHOR]

In biomedical applications, nanomaterial-based delivery vehicles, such as lipid nanoparticles, have emerged as promising instruments for improving the solubility, stability, and encapsulation of various payloads. This article provides a formal review focusing on the reactogenicity of empty lipid nanoparticles used as delivery vehicles, specifically emphasizing their application in mRNA-based therapies. Reactogenicity refers to the adverse immune responses triggered by xenobiotics, including administered lipid nanoparticles, which can lead to undesirable therapeutic outcomes. The key components of lipid nanoparticles, which include ionizable lipids and PEG-lipids, have been identified as significant contributors to their reactogenicity. Therefore, understanding the relationship between lipid nanoparticles, their structural constituents, cytokine production, and resultant reactogenic outcomes is essential to ensure the safe and effective application of lipid nanoparticles in mRNA-based therapies. Although efforts have been made to minimize these adverse reactions, further research and standardization are imperative. By closely monitoring cytokine profiles and assessing reactogenic manifestations through preclinical and clinical studies, researchers can gain valuable insights into the reactogenic effects of lipid nanoparticles and develop strategies to mitigate undesirable reactions. This comprehensive review underscores the importance of investigating lipid nanoparticle reactogenicity and its implications for the development of mRNA–lipid nanoparticle therapeutics in various applications beyond vaccine development. [ABSTRACT FROM AUTHOR]

Therapeutics based on messenger RNA (mRNA) have become a promising class of methods for various diseases' treatments in recent years. The safe and effective delivery of mRNA into target cells is the premise of the clinical translation of mRNA. Due to the protonation of ionizable lipids in acidic conditions, the ionizable lipid nanoparticles (iLNPs) have lower toxicity and higher endosomal escape efficiency than permanent cationic lipid nanoparticles (cLNPs). They not only exhibit excellent delivery ability in commercial COVID-19 mRNA vaccines but also are highly potential carriers for delivering mRNA drugs. This review summarizes the composition, preparation, stability of mRNA iLNPs, and the recent progress in the mRNA delivery by iLNPs. According to the structure characteristics of ionizable heads, the ionizable lipids are classified into tertiary amine, quaternary amine, imidazole core, piperidine core, piperazine core, and diketopiperazine core-based head lipids. Their design, optimization, and applications on mRNA delivery are discussed. The current status of representative clinical trials based on mRNA iLNPs delivery is also analyzed. The challenges and prospects of mRNA iLNPs delivery in clinical applications are proposed at the end. [ABSTRACT FROM AUTHOR]

The article offers a review of U.S. Pharmacopeia (USP) chapters relating to the stability, efficacy and safety of messenger RNA (mRNA)-based lipid nanoparticle (LNP)-based COVID-19 vaccines. Topics discussed include the control of particulate matter in injections, particle size distribution (PSD) in lipid injectable emulsions, and technology for quantifying the critical large-diameter tail (LDT) region in PSD.

Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near‐infrared (NIR‐II) lipid nanoparticles (LNPs) containing pH activatable NIR‐II dye conjugated lipid (Cy‐lipid) for potentiating mRNA delivery efficiency via a stimulus‐responsive photothermal‐promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy‐lipid is protonated and turns on NIR‐II absorption for light‐to‐heat transduction mediated by 1064 nm laser irradiation. Then, the heat‐promoted LNPs morphology change triggers rapid escape of NIR‐II LNPs from the endosome, allowing about 3‐fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR‐II light free group. In addition, the bioluminescence intensity induced by delivered luciferase encoding mRNA in the mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy. [ABSTRACT FROM AUTHOR]

Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near‐infrared (NIR‐II) lipid nanoparticles (LNPs) containing pH activatable NIR‐II dye conjugated lipid (Cy‐lipid) for potentiating mRNA delivery efficiency via a stimulus‐responsive photothermal‐promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy‐lipid is protonated and turns on NIR‐II absorption for light‐to‐heat transduction mediated by 1064 nm laser irradiation. Then, the heat‐promoted LNPs morphology change triggers rapid escape of NIR‐II LNPs from the endosome, allowing about 3‐fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR‐II light free group. In addition, the bioluminescence intensity induced by delivered luciferase encoding mRNA in the mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy. [ABSTRACT FROM AUTHOR]

Ionizable lipid-containing lipid nanoparticles (LNPs) as a non-viral vector with good safety and potency have been considered as an ideal delivery system for gene therapy. The screening of ionizable lipid libraries with common features but diverse structures holds the promise of finding new candidates for LNPs to deliver different nucleic acid drugs such as messenger RNAs (mRNAs). Chemical strategies for the facile construction of ionizable lipid libraries with diverse structure are in high demand. Here, we report on the ionizable lipids containing the triazole moiety prepared by the copper-catalyzed alkyne–azide click reaction (CuAAC). We demonstrated that these lipids served well as the major component of LNPs, in order to encapsulate mRNA using luciferase mRNA as the model system. Thus, this study shows the potential of click chemistry in the preparation of lipid libraries for LNP assembly and mRNA delivery. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer is one of the most common cancers worldwide, and novel treatments are urgently needed to improve treatment for cancer patients. In this report, three different designs of humanized EpCAM-CD3 antibodies were engineered and tested against colorectal tumors. The antibodies demonstrated high efficacy and specificity. In addition, the study demonstrates a novel method of delivering bispecific antibodies using mRNA-lipid nanoparticle (LNP) technology. The delivery of EpCAM-CD3 human Fc (hFc) mRNA-LNPs into mice tumors with intravenous T cell injection significantly blocked OVCAR-5 xenograft tumor growth in vivo. The data provide a basis for future clinical studies. The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) lipid nanoparticles (LNPs) have emerged at the forefront during the COVID-19 vaccination campaign. Despite their tremendous success, mRNA vaccines currently require storage at deep freeze temperatures which complicates their storage and distribution, and ultimately leads to lower accessibility to low- and middle-income countries. To elaborate on this challenge, we investigated freeze-drying as a method to enable storage of mRNA LNPs at room- and even higher temperatures. More specifically, we explored a novel continuous freeze-drying technique based on spin-freezing, which has several advantages compared to classical batch freeze-drying including a much shorter drying time and improved process and product quality controlling. Here, we give insight into the variables that play a role during freeze-drying by evaluating the impact of the buffer and mRNA LNP formulation (ionizable lipid to mRNA weight ratio) on properties such as size, morphology and mRNA encapsulation. We found that a sufficiently high ionizable lipid to mRNA weight ratio was necessary to prevent leakage of mRNA during freeze-drying and that phosphate and Tris, but not PBS, were appropriate buffers for lyophilization of mRNA LNPs. We also studied the stability of optimally lyophilized mRNA LNPs at 4 °C, 22 °C, and 37 °C and found that transfection properties of lyophilized mRNA LNPs were maintained during at least 12 weeks. To our knowledge, this is the first study that demonstrates that optimally lyophilized mRNA LNPs can be safely stored at higher temperatures for months without losing their transfection properties. [Display omitted] • Continuous freeze-drying is suitable for the lyophilization of mRNA LNPs. • Lipid:mRNA ratio and type of buffer are crucial in lyophilization of mRNA LNPs. • Formulation buffer affects mRNA LNP morphology upon formation and lyophilization. • Lyophilization in Tris buffer enhances mRNA LNP stability at higher temperatures. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID‐19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF‐A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF‐A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP‐mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF‐A mRNA delivery to cells via LNPs, a fraction of internalized VEGF‐A mRNA is secreted via EVs. The overexpressed VEGF‐A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA‐Seq analysis reveals that as cells' response to LNP‐VEGF‐A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF‐A mRNA in vitro and in vivo. Upon equal amount of VEGF‐A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF‐A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF‐A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF‐A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs. [ABSTRACT FROM AUTHOR]

High-fat and high-salt diets are risk factors for metabolic syndrome development. However, gochujang, which has a high salt content, possesses antiobesity properties in cell and animal models. We aimed to evaluate the effects of Sunchang traditional and modern factory produced gochujang on metabolic syndrome factors in high-fat diet (HFD)-induced obese mice. For 14 weeks, 4-week-old C57BL/6J male mice were separated into five groups and fed a normal diet (ND), a high-fat diet only (HD), a HD with salt (SALT), a HD with traditional Sunchang gochujang (TS), and HD with modern factory made Sunchang gochujang (FS). Compared to HD and SALT groups, the gochujang groups had lower body weight, blood leptin, and insulin levels with reduced Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index and improved serum and liver lipid profiles. In addition, gochujang supplemented groups exhibited a significant reduction in mRNA expression of anabolic lipid metabolism related factors; PPARγ, CEBPα, and FABP4, and a significant increase in mRNA expression of energy expenditure-related factors; PPARα and CPT1. Protein expressions of SREBP1 were downregulated in the gochujang fed groups. TS and FS intakes improved obesity in HFD-induced obese mice. Compared to the gochujang groups, the SALT group did not exhibit any of those benefits suggesting that the high salt content of gochujang has different effects compared with added salt alone. Our findings provide evidence that gochujang could be a functional food to attenuate metabolic syndrome. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g. , induction of antigen specific tolerance), or within sensitive tissues (e.g. , central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (M n = 2 k, 5 k) and anchor diacyl chain length (m = 14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS. [Display omitted] [ABSTRACT FROM AUTHOR]

Statins are the cornerstone of lipid-lowering therapies effective for cardiovascular risk reduction. Although they are generally well tolerated, statin intolerance (SI) is frequent in clinical practice, and it is usually related to the onset of muscle symptoms, which are defined under the acronym SAMS (Statin-Associated Muscle Side Effects). These side effects are responsible for statin treatment discontinuation that results in increased cardiovascular risk. The National Lipid Association (NLA) has recently provided an updated definition of statin intolerance, and a distinction between complete and partial statin intolerance has been reported. The evaluation of symptom severity and the presence of muscle damage biomarker alterations make it essential to adopt a patient-centered approach aimed at obtaining a personalized therapeutic strategy. Firstly, it could be useful to administer a different statin, reduce the dosage or adopt an alternate dosage regimen. However, some patients are unable to tolerate any statin at every dosage, or despite taking statins at the maximum tolerated dose, they fail to achieve the recommended LDL-C target, and thus it is necessary to introduce a non-statin hypolipidemic treatment. Ezetimibe, proprotein-convertase subtilisin/kexin type 9 (PCSK9) inhibitors such as monoclonal antibodies (alirocumab and evolocumab) or RNA messenger silencing (inclisiran), bempedoic acid or nutraceuticals are non-statin lipid-lowering therapies that could be used as an alternative or in addition to statins to achieve an early and sustained LDL-C reduction in clinical practice. In this review, we evaluated SI management focusing on non-statin lipid lowering therapies and their implications in lipid lowering approaches in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD), a type of neurodegeneration disease, is characterized by Aβ deposition and tangles of nerve fibers. Schisandrin is one of the main components of Fructus Schisandrae Chinensis. Researches showed that schisandrin can improve the cognitive impairment and memory of AD mice, but the specific mechanism has not been fully elucidated. Purpose: The purpose of this study is to investigate the possible mechanism of schisandrin in improving AD pathology. Methods: The Morris water maze test was executed to detect spatial learning and memory. Ultra performance liquid chromatography-Triple time of flight mass spectrometry (UPLC-Triple-TOF/MS)-based plasma lipidomics was used to study the changes of plasma lipids. Moreover, we measured the levels of protein and mRNA expression of APOE and ABCA1 in the rat brains and in BV2 microglia. Results: Our study found that schisandrin could improve learning and memory, and reduce Aβ deposition in AD rats. Furthermore, we found that schisandrin can improve plasma lipid metabolism disorders. Therefore, we hypothesized schisandrin might act via LXR and the docking results showed that schisandrin interacts with LXRβ. Further, we found schisandrin increased the protein and mRNA expression of LXR target genes APOE and ABCA1 in the brain of AD rats and in BV2 microglia. Conclusion: Our study reveals the neuroprotective effect and mechanism of schisandrin improves AD pathology by activating LXR to produce APOE and ABCA1. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are the commonly used delivery tools for messenger RNA (mRNA) therapy and play an indispensable role in the success of COVID-19 mRNA vaccines. Ionizable cationic lipids are the most important component in LNPs. Herein, we developed a series of new ionizable lipids featuring bioreducible disulfide bonds, and constructed a library of lipids derived from dimercaprol. LNPs prepared from these ionizable lipids could be stored at 4 °C for a long term and are non-toxic toward HepG2 and 293T cells. In vivo experiments demonstrated that the best C4S18A formulations, which embody linoleoyl tails, show strong firefly luciferase (Fluc) mRNA expression in the liver and spleen via intravenous (IV) injection, or at the local injection site via intramuscular injection (IM). The newly designed ionizable lipids can be potentially safe and high-efficiency nanomaterials for mRNA therapy. [ABSTRACT FROM AUTHOR]

In the present study, we analyzed the patent application status and progress of lipid nanoparticle (LNP) delivery technology for mRNA vaccines on a global scale, focusing on the technological development dynamics. Moreover, we compared the relevant situation of domestic and foreign applicants, aiming to provide references for researchers engaged in mRNA vaccine delivery systems in the domestic pharmaceutical field. [ABSTRACT FROM AUTHOR]

We have examined the effect of gypenosides on hepatic lipid metabolism in hyperlipidemic mice. To this end, male mice were placed on a high-fat diet and then administered gypenosides. Compared to the mice on the high-fat diet, the levels of liver index, total cholesterol, and low-density lipoprotein cholesterol in the serum were significantly reduced by gypenoside treatment. Gypenosides influenced the overall profile of 22 hepatic fatty acid metabolism genes significantly. Three genes, Cyp4a10, Cyp4a14, and Cyp4a31, had their mRNA expression altered by about tenfold. As a possible mechanism, the findings suggest that gypenosides regulate lipid homeostasis by influencing fatty acid metabolism and promoting fatty acid ω-oxidation. [ABSTRACT FROM AUTHOR]

The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax. [ABSTRACT FROM AUTHOR]

Purpose: Messenger RNA (mRNA) has shown great promise for vaccine against both infectious diseases and cancer. However, mRNA is unstable and requires a delivery vehicle for efficient cellular uptake and degradation protection. So far, lipid nanoparticles (LNPs) represent the most advanced delivery platform for mRNA delivery. However, no published studies have compared lipid microparticles (LMPs) with lipid nanoparticles (LNPs) in delivering mRNA systematically, therefore, we compared the impact of particle size on delivery efficacy of mRNA vaccine and subsequent immune responses. Methods: Herein, we prepared 3 different size lipid particles, from nano-sized to micro-sized, and they loaded similar amounts of mRNA. These lipid particles were investigated both in vitro and in vivo, followed by evaluating the impact of particle size on inducing cellular and humoral immune responses. Results: In this study, all mRNA vaccines showed a robust immune response and lipid microparticles (LMPs) show similar efficacy with lipid nanoparticles (LNPs) in delivering mRNA and preventing cancer. In addition, immune adjuvants, either toll like receptors or active molecules from traditional Chinese medicine, can improve the efficacy of mRNA vaccines. Conclusions: Considering the efficiency of delivery and endocytosis, besides lipid nanoparticles with size smaller than 150 nm, lipid microparticles (LMPs) also have the potential to be an alternative and promising delivery system for mRNA vaccines. [ABSTRACT FROM AUTHOR]