pH-Dependent Phase Behavior and Stability of Cationic Lipid–mRNA Nanoparticles

Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior.The mRNA–LNPs transition from an inverse hexagonal phase at pH values below the pK_a of the cationic lipid to a lamellar phase above the pK_a. At higher temperatures the mRNA–LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pK_a than above it.Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA–LNPs. The particles were larger and more prone to aggregation below the pK_a. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA–LNPs had the highest transfection efficiency and stability at pH values below the pK_a. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA–LNPs and will aid the development of related formulations.     

Lipid-based nanoparticle formulations for small molecules and RNA drugs

ABSTRACT

Introduction: Liposomes and lipid-based nanoparticles (LNPs) effectively deliver cargo molecules to specific tissues, cells, and cellular compartments. Patients benefit from these nanoparticle formulations by altered pharmacokinetic properties, higher efficacy, or reduced side effects. While liposomes are an established delivery option for small molecules, Onpattro® (Sanofi Genzyme, Cambridge, MA) is the first commercially available LNP formulation of a small interfering ribonucleic acid (siRNA).

Areas covered: This review article summarizes key features of liposomal formulations for small molecule drugs and LNP formulations for RNA therapeutics. We describe liposomal formulations that are commercially available or in late-stage clinical development and the most promising LNP formulations for ASOs, siRNAs, saRNA, and mRNA therapeutics.

Expert opinion: Similar to liposomes, LNPs for RNA therapeutics have matured but still possess a niche application status. RNA therapeutics, however, bear an immense hope for difficult to treat diseases and fuel the imagination for further applications of RNA drugs. LNPs face similar challenges as liposomes including limitations in biodistribution, the risk to provoke immune responses, and other toxicities. However, since properties of RNA molecules within the same group are very similar, the entire class of therapeutic molecules would benefit from improvements in a few key parameters of the delivery technology.     

Characterization of polysaccharides and their antioxidant properties from Plumula nelumbinis

Two novel polysaccharides, Plumula nelumbinis (P. nelumbinis) polysaccharide I (LNP I) and P. nelumbinis polysaccharide II (LNP II), were extracted and purified from P. nelumbinis, and a sulfated polysaccharide, P. nelumbinis polysaccharide III (LNP III), with a substitution degree of 0.62 was prepared from LNPI. The structures of the LNPs were preliminarily characterized using high performance size exclusion chromatography (HPSEC), gas chromatography-mass spectrometry (GC–MS), Fourier transformed infrared spectrometry (FT-IR), and nuclear magnetic resonance (NMR) spectrometry. In addition, evaluation of the antioxidant activity of the LNPs showed that they could significantly increase the proliferation of RAW264.7 macrophages (P?<?0.05) and improve the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) based on cell model of H₂O_2-induced oxidative damage. This suggested that these LNPs may be used as potential antioxidants.     

Tailoring combinatorial lipid nanoparticles for intracellular delivery of nucleic acids,proteins, and drugs

Lipid nanoparticle (LNP)-based drug delivery systems have become the most clinically advanced non-viral delivery technology. LNPs can encapsulate and deliver a wide variety of bioactive agents, including the small molecule drugs, proteins and peptides, and nucleic acids. However, as the physicochemical properties of small- and macromolecular cargos can vary drastically, every LNP carrier system needs to be carefully tailored in order to deliver the cargo molecules in a safe and efficient manner. Our group applied the combinatorial library synthesis approach and in vitro and in vivo screening strategy for the development of LNP delivery systems for drug delivery. In this Review, we highlight our recent progress in the design, synthesis, characterization, evaluation, and optimization of combinatorial LNPs with novel structures and properties for the delivery of small- and macromolecular therapeutics both in vitro and in vivo. These delivery systems have enormous potentials for cancer therapy, antimicrobial applications, gene silencing, genome editing, and more. We also discuss the key challenges to the mechanistic study and clinical translation of new LNP-enabled therapeutics.     

Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA

Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na⁺/K⁺ ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency.From the Clinical EditorIn this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency.     

Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA

The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species. In particular, the low potency of LNP containing DLinDAP can be attributed to hydrolysis by endogenous lipases following uptake. LNP containing DLinKC2-DMA, DLinKDMA, or DLinDMA, which lack ester linkages, are not vulnerable to lipase digestion and facilitate much more potent gene silencing. The superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to higher uptake and improved ability to stimulate siRNA release from endosomes subsequent to uptake.From the Clinical EditorThis study reports on the in vivo gene silencing potency of lipid nanoparticle-siRNA systems containing ionizable cationic lipids. It is concluded that the superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to their higher uptake thus improved ability to stimulate siRNA release from endosome.     

Lipid nanoparticle purification by spin centrifugation-dialysis (SCD): a facile and high-throughput approach for small scale preparation of siRNA-lipid complexes

This paper describes the use of Spin Centrifugation-Dialysis (SCD) for small-scale concentration/purification of siRNA-lipid complexes designed for use as therapeutic agents for gene silencing. SCD consists of a two-step method for concentration, filtration and buffer exchange of Lipid Nanoparticles (LNP) to provide a homogeneous preparation suitable for injection. Here, we compare SCD with the more traditionally used Tangential Flow Filtration (TFF), and demonstrate the physicochemical and biological comparability of LNPs produced with both methods. TFF is a highly scalable method used in both developmental and production applications, but is limited in terms of miniaturization. In contrast to TFF, SCD is faster, less expensive, and requires less oversight for assembling LNPs for small-scale applications, such as target screening both in vitro and in vivo. The finding that SCD is a viable method for filtering LNPs in a manner similar to TFF, producing particles with comparable properties and biological activity, is significant given the complexity and sensitivity of LNPs to processing conditions.     

Lipid nanoparticles: state of the art,new preparation methods and challenges in drug delivery

Introduction: Nanoparticles are rapidly developing as drug carriers because of their size-dependent properties. Lipid nanoparticles (LNPs) are widely employed in drug delivery because of the biocompatibility of the lipid matrix.

Areas covered: Many different types of LNPs have been engineered in the last 20 years, the most important being solid lipid nanoparticles (SLNs), nanostrucured lipid carriers (NLCs), lipid–drug conjugates (LDCs) and lipid nanocapsules (LNCs). This review gives an overview of LNPs, including their physico-chemical properties and pharmacological uses. Moreover, it highlights the most important innovations in the preparation techniques of LNPs, aimed to encapsulate different molecules within the lipid matrix. Finally, it gives a short perspective on the challenges of drug delivery, which are a potential field of application for LNPs: cancer therapy, overcoming the blood–brain barrier and gene and protein delivery.

Expert opinion: LNPs are a safe and versatile vehicles for drug and active delivery, suitable for different administration routes. New technologies have been developed for LNP preparation and studies are currently underway in order to obtain the encapsulation of different drugs and to deliver the active molecule to the site of action.     

The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles.     

Single pot organic solvent-free thermocycling technology for siRNA-ionizable LNPs: a proof-of-concept approach for alternative to microfluidics

Ionizable LNPs are the latest trend in nucleic acid delivery. Microfluidics technology has recently gained interest owing to its rapid mixing, production of nucleic acid-ionizable LNPs, and stability of nucleic acid inside the body. Industrial scale-up, nucleic acid-lipid long-term storage instability, and high production costs prompted scientists to seek alternate solutions to replace microfluidic technology. We proposed a single-pot, organic solvent-free thermocycling technology to efficiently and economically overcome most of the limitations of microfluidic technology. New thermocycling technology needs optimization of process parameters such as sonication duration, cooling–heating cycle, number of thermal cycles, and lipid:aqueous phase ratio to formulate precisely sized particles, effective nucleic acid encapsulation, and better shelf-life stability. Our research led to the formulation of siRNA-ionizable LNPs with particle sizes of 104.2 ± 34.7 nm and PDI 0.111 ± 0.109, with 83.3 ± 4.1% siRNA encapsulation. Thermocycling siRNA-ionizable LNPs had comparable morphological structures with commercialized microfluidics ionizable LNPs imaged by TEM and cryo-TEM. When compared to microfluidics ionizable LNPs, thermocycling siRNA-ionizable LNPs had a longer shelf life at 4°C. Our thermocycling technology showed an effective alternative to microfluidics technology in the production of nucleic acid–ionizable LNPs to meet global demand.     

Lipid nanoparticles: state of the art, new preparation methods and challenges in drug delivery

INTRODUCTION: Nanoparticles are rapidly developing as drug carriers because of their size-dependent properties. Lipid nanoparticles (LNPs) are widely employed in drug delivery because of the biocompatibility of the lipid matrix. AREAS COVERED: Many different types of LNPs have been engineered in the last 20 years, the most important being solid lipid nanoparticles (SLNs), nanostrucured lipid carriers (NLCs), lipid-drug conjugates (LDCs) and lipid nanocapsules (LNCs). This review gives an overview of LNPs, including their physico-chemical properties and pharmacological uses. Moreover, it highlights the most important innovations in the preparation techniques of LNPs, aimed to encapsulate different molecules within the lipid matrix. Finally, it gives a short perspective on the challenges of drug delivery, which are a potential field of application for LNPs: cancer therapy, overcoming the blood-brain barrier and gene and protein delivery. EXPERT OPINION: LNPs are a safe and versatile vehicles for drug and active delivery, suitable for different administration routes. New technologies have been developed for LNP preparation and studies are currently underway in order to obtain the encapsulation of different drugs and to deliver the active molecule to the site of action.     

Characterization of lipid nanoparticles by differential scanning calorimetry, X-ray and neutron scattering

Differential scanning calorimetry and X-ray diffraction play a prominent role in the characterization of lipid nanoparticle (LNP) dispersions. This review shortly outlines the measurement principles of these two techniques and summarizes their applications in the field of nanodispersions of solid lipids. These methods are particularly useful for the characterization of the matrix state, polymorphism and phase behavior of the nanoparticles which may be affected by, for example, the small particle size and the composition of the dispersions. The basics of small angle X-ray and neutron scattering which are also very promising methods for the characterization of LNPs are explained in some more detail. Examples for their use in the area of solid LNPs regarding the evaluation of particle size effects and the formation of superstructures in the nanoparticle dispersions are given. Some technical questions concerning the use of the different characterization techniques in the field of LNP research are also addressed.     

Synergistic Enhancement of Cellular Uptake With CD44-Expressing Malignant Pleural Mesothelioma by Combining Cationic Liposome and Hyaluronic Acid–Lipid Conjugate

Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44.     

Predicting liposome formulations by the integrated machine learning and molecular modeling approaches

Liposome is one of the most widely used carriers for drug delivery because of the great biocompatibility and biodegradability. Due to the complex formulation components and preparation process, formulation screening mostly relies on trial-and-error process with low efficiency. Here liposome formulation prediction models have been built by machine learning (ML) approaches. The important parameters of liposomes, including size, polydispersity index (PDI), zeta potential and encapsulation, are predicted individually by optimal ML algorithm, while the formulation features are also ranked to provide important guidance for formulation design. The analysis of key parameter reveals that drug molecules with logS [-3, -6], molecular complexity [500, 1000] and XLogP3 (≥2) are priority for preparing liposome with higher encapsulation. In addition, naproxen (NAP) and palmatine HCl (PAL) represented the insoluble and water-soluble molecules are prepared as liposome formulations to validate prediction ability. The consistency between predicted and experimental value verifies the satisfied accuracy of ML models. As the drug properties are critical for liposome particles, the molecular interactions and dynamics of NAP and PAL liposome are further investigated by coarse-grained molecular dynamics simulations. The modeling structure reveals that NAP molecules could distribute into lipid layer, while most PAL molecules aggregate in the inner aqueous phase of liposome. The completely different physical state of NAP and PAL confirms the importance of drug properties for liposome formulations. In summary, the general prediction models are built to predict liposome formulations, and the impacts of key factors are analyzed by combing ML with molecular modeling. The availability and rationality of these intelligent prediction systems have been proved in this study, which could be applied for liposome formulation development in the future.     

对世界卫生组织预防传染病mRNA疫苗非临床评价技术要点的解析

目的：了解WHO最新发布的《预防传染病mRNA疫苗质量、安全及有效性评价法规考虑》中关于mRNA疫苗非临床评价的主要内容,为我国评价此类产品非临床研究提供参考。方法：分析mRNA疫苗的主要特点,对照《预防传染病mRNA疫苗质量、安全及有效性评价法规考虑》,梳理mRNA疫苗非临床研究特别是药效学和毒理学研究设计、实施和分析的关键要点。结果与结论：mRNA技术已成为疫苗研发的前沿技术,作为新型生物制品,mRNA疫苗具有诸多不同于传统生物制品的特点。WHO认为非临床研究的设计、实施和分析应充分考虑mRNA产品的技术特点,相关药效学和毒理学研究应着重解决免疫应答的持久性或持久性免疫细胞表型、Ⅰ型干扰素介导的固有免疫应答、mRNA和LNPs的生物分布和持久性、全身和局部的毒性和炎症反应、非自然修饰核苷的潜在毒性、脂质纳米颗粒中新型脂质的毒性等方面问题,并提出了在公共卫生突发事件背景下针对优先病原体的mRNA疫苗非临床加速评价的考虑要点,对我国非临床评价此类产品具有很好的指导作用和应用价值。     

Design and biological activity of novel stealth polymeric lipid nanoparticles for enhanced delivery of hydrophobic photodynamic therapy drugs

Advances in in vivo stability and preferential tumor uptake of cancer nanomedicine are warranted for effective chemotherapy. Here, we describe a novel nanoformulation using an unconventional polymeric tubule-forming phospholipid, DC_8,9PC. We report that DC_8,9PC transitions to stable vesicles (LNPs) in the presence of PEGylated lipid (DSPE-PEG2000); the resulting DC_8,9PC:DSPE-PEG2000 LNPs efficiently included a hydrophobic PDT drug, HPPH. Remarkably, these LNPs incorporated unusually high DSPE-PEG2000 concentrations; LNP₁₀-HPPH and LNP₂₀-HPPH (10 & 20 mol% PEGylated lipid, respectively) exhibited >90% serum stability at 37?°C. Increased PEGylation in the LNPs correlated with enhanced tumor accumulation in intravenously injected HT29 tumor mouse xenographs. Colon-26 bearing BALB/c mice, intravenously injected with LNP₂₀-HPPH showed superior PDT efficacy and animal survival (no tumor recurrence up to 100 days) as compared to a formulation currently used in clinical trials. Taken together, we present a simple stealth binary lipid nanosystem with enhanced efficiency of tumor accumulation and superior therapeutic efficacy.     

Effect of PEGylation on Biodistribution and Gene Silencing of siRNA/Lipid Nanoparticle Complexes

Purpose

To determine the influence of physicochemical properties of lipid nanoparticles (LNPs) carrying siRNA on their gene silencing in vivo. Mechanistic understanding of how the architecture of the nanoparticle can alter gene expression has also been studied.

Methods

The effect of 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA) on hepatic distribution and FVII gene silencing was determined. FVII mRNA in hepatocytes and liver tissues was determined by Q-PCR. Hepatic distribution was quantified by FACS analysis using Cy5 labeled siRNA.

Results

Gene silencing was highly dependent on the amount of PEG-C-DMA present. FVII gene silencing inversely correlated to the amount of PEG-C-DMA in LNPs. High FVII gene silencing was obtained in vitro and in vivo when the molar ratio of PEG-C-DMA to lipid was 0.5 mol%. Surprisingly, PEGylation didn’t alter the hepatic distribution of the LNPs at 5 h post administration. Instead the amount of PEG present in the LNPs has an effect on red blood cell disruption at low pH.

Conclusion

Low but sufficient PEG-C-DMA amount in LNPs plays an important role for efficient FVII gene silencing in vivo. PEGylation did not alter the hepatic distribution of LNPs, but altered gene silencing efficacy by potentially reducing endosomal disruption.     

Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran) and mRNA (COVID-19 vaccine) drugs

Nucleic acid therapeutics are developing into precise medicines that can manipulate specific genes. However, the development of safe and effective delivery system for the target cells has remained a challenge. Lipid nanoparticles (LNPs) have provided a revolutionary delivery system that can ensure multiple clinical translation of RNA-based candidates. In 2018, Patisiran (Onpattro) was first approved as an LNP-based siRNA drug. In 2020, during the coronavirus disease 2019 (COVID-19) outbreak, LNPs have enabled the development of two SARS-CoV-2 mRNA vaccines, Tozinameran (Comirnaty or Pfizer-BioNTech COVID-19 vaccine) and Elasomeran (Spikevax or COVID-19 vaccine Moderna) for conditional approval. Here, we reviewed the state-of-the-art LNP technology employed in three approved drugs (one siRNA-based and two mRNA-based drugs) and discussed the differences in their mode of action, formulation design, and biodistribution.     

Feature importance of machine learning prediction models shows structurally active part and important physicochemical features in drug design

The objective of this study was to obtain the indicators of physicochemical parameters and structurally active sites to design new chemical entities with desirable pharmacokinetic profiles by investigating the process by which machine learning prediction models arrive at their decisions, which are called explainable artificial intelligence. First, we developed the prediction models for metabolic stability, CYP inhibition, and P-gp and BCRP substrate recognition using 265 physicochemical parameters for designing the molecular structures. Four important parameters, including the well-known indicator h_logD, are common in some in vitro studies; as such, these can be used to optimize compounds simultaneously to address multiple pharmacokinetic concerns. Next, we developed machine learning models that had been programmed to show structurally active sites. Many types of machine learning models were developed using the results of in vitro metabolic stability study of around 30000 in-house compounds. The metabolic sites of in-house compounds predicted using some prediction models matched experimentally identified metabolically active sites, with a ratio of number of metabolic sites (predicted/actual) of over 90%. These models can be applied to several screening projects. These two approaches can be employed for obtaining lead compounds with desirable pharmacokinetic profiles efficiently.     

Recent advances in lipid nanoparticles for delivery of nucleic acid,mRNA, and gene editing-based therapeutics

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.