Helper Lipid Research Articles

Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

PH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model.The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Х3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Х3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

Bile acid-containing lipid nanoparticles enhance extrahepatic mRNA delivery.

Lipid nanoparticles (LNPs) have emerged as a viable, clinically-validated platform for the delivery of mRNA therapeutics. LNPs have been utilized as mRNA delivery systems for applications including vaccines, gene therapy, and cancer immunotherapy. However, LNPs, which are typically composed of ionizable lipids, cholesterol, helper lipids, and lipid-anchored polyethylene glycol, often traffic to the liver which limits the therapeutic potential of the platform. Several approaches have been proposed to resolve this tropism such as post-synthesis surface modification or the addition of synthetic cationic lipids. Methods: Here, we present a strategy for achieving extrahepatic delivery of mRNA involving the incorporation of bile acids, a naturally-occurring class of cholesterol analogs, during LNP synthesis. We synthesized a series of bile acid-containing C14-4 LNPs by replacing cholesterol with bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid) at various ratios. Results: Bile acid-containing LNPs (BA-LNPs) were able to reduce delivery to liver cells in vitro and improve delivery in a variety of other cell types, including T cells, B cells, and epithelial cells. Our subsequent in vivo screening of selected LNP candidates injected intraperitoneally or intravenously identified a highly spleen tropic BA-LNP: CA-100, a four-component LNP containing cholic acid and no cholesterol. These screens also identified BA-LNP candidates demonstrating promise for other mRNA therapeutic applications such as for gastrointestinal or immune cell delivery. We further found that the substitution of cholic acid for cholesterol in an LNP formulation utilizing a different ionizable lipid, C12-200, also shifted mRNA delivery from the liver to the spleen, suggesting that this cholic acid replacement strategy may be generalizable. Conclusion: These results demonstrate the potential of a four-component BA-LNP formulation, CA-100, for extrahepatic mRNA delivery that could potentially be utilized for a range of therapeutic and vaccine applications.

Navigating the challenges of lipid nanoparticle formulation: the role of unpegylated lipid surfactants in enhancing drug loading and stability.

Lipid nanoparticles have proved an attractive approach for drug delivery; however, the challenges of optimising formulation stability and increasing drug loading have limited progression. In this work, we investigate the role of unpegylated lipid surfactants (helper lipids) in nanoparticle formation and the effect of blending helper lipids with pegylated lipid surfactants on the formation and stability of lipid-based nanoparticles by nanoprecipitation. Furthermore, blends of unpegylated/pegylated lipid surfactants were examined for ability to accommodate higher drug loading formulations by means of a higher weight percentage (wt%) of drug relative to total mass of formulation components (i.e. drug, surfactants and lipids). Characterisation included evaluation of particle diameter, size distribution, drug loading and nanoformulation stability. Our findings demonstrate that the addition of unpegylated lipid surfactant (Lipoid S100) to pegylated lipid surfactant (Brij S20) enhances stability, particularly at higher weight percentages of the core material. This blending approach enables drug loading capacities exceeding 10% in the lipid nanoparticles. Notably, Lipoid S100 exhibited nucleating properties that aided in the formation and stabilisation of the nanoparticles. Furthermore, we examined the incorporation of a model drug into the lipid nanoparticle formulations. Blending the model drug with the core material disrupted the crystallinity of the core, offering additional potential benefits in terms of drug release and stability. This comprehensive investigation provides valuable insights into the interplay between surfactant properties, core material composition, and nanoparticle behaviour. The study enhances our understanding of lipid materials and offers guidance for the design and optimisation of lipid nanoparticle formulations.

Formulation of lipid nanoparticles containing ginsenoside Rg2 and protopanaxadiol for highly efficient delivery of mRNA.

Lipid nanoparticles (LNPs) are widely recognized as crucial carriers of mRNA in therapeutic and vaccine development. The typical lipid composition of mRNA-LNP systems includes an ionizable lipid, a helper lipid, a polyethylene glycol (PEG)-lipid, and cholesterol. Concerns arise regarding cholesterol's susceptibility to oxidation, potentially leading to undesired immunological responses and toxicity. In this study, we formulated novel LNPs by replacing cholesterol with phytochemical-derived compounds, specifically ginsenoside Rg2 and its derivative phytosterol protopanaxadiol (PPD), and validated their efficacy as mRNA delivery systems. The mRNA-LNP complexes were manually prepared through a simple mixing process. The biocompatibility of these Rg2-based LNPs (Rg2-LNP) and PPD-based LNPs (PPD-LNP) was assessed through cell viability assays, while the protective function of LNPs for mRNA was demonstrated by RNase treatment. Enhanced green fluorescent protein (EGFP) mRNA delivery and expression in A549 and HeLa cells were analyzed using optical microscopy and flow cytometry. The expression efficiency of Rg2-LNP and PPD-LNP was compared with that of commercially available LNPs, with both novel formulations demonstrating superior transfection and EGFP expression. Furthermore, in vivo tests following intramuscular (I.M.) injection in hairless mice demonstrated efficient luciferase (Luc) mRNA delivery and effective Luc expression using Rg2-LNP and PPD-LNP compared to commercial LNPs. Results indicated that the efficiency of EGFP and Luc expression in Rg2-LNP and PPD-LNP surpassed that of the cholesterol-based LNP formulation. These findings suggest that Rg2-LNP and PPD-LNP are promising candidates for future drug and gene delivery systems.

Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

Development of a Ready-to-Use-Type RNA Vaccine Carrier Based on an Intracellular Environment-Responsive Lipid-like Material with Immune-Activating Vitamin E Scaffolds.

Because of its efficient and robust gene transfer capability, messenger RNA (mRNA) has become a promising tool in various research fields. The lipid nanoparticle (LNP) is considered to be a fundamental technology for an mRNA delivery system and has been used extensively for the development of RNA vaccines against SARS-CoV-2. We recently developed ssPalm, an environmentally responsive lipid-like material, as a component of LNP for mRNA delivery. In this study, a self-degradable unit (phenyl ester) that confers high transfection activity and an immune stimulating unit (vitamin E scaffold) for high immune activation were combined to design a material, namely, ssPalmE-Phe-P4C2, for vaccine use. To design a simple and user-friendly form of an RNA vaccine based on this material, a freeze-drying-based preparation method for producing a ready-to-use-type LNP (LNP(RtoU)) was used to prepare the LNPssPalmE-Phe. The optimization of the preparation method and the lipid composition of the LNPssPalmE-Phe(RtoU) revealed that dioleoyl-sn-glycero phosphatidylethanolamine (DOPE) was a suitable helper lipid for achieving a high vaccination activity of the LNPssPalmE-Phe(RtoU). Other findings indicated that to maintain particle properties and vaccination activity, a 40% cholesterol content was necessary. A single administration of the LNPssPalmE-Phe(RtoU) that contained mRNA-encoding Ovalbumin (mOVA-LNPssPalmE-Phe(RtoU)) demonstrated a significant suppression of tumor progression in a tumor-bearing mouse OVA-expressing cell line (E.G7-OVA). In summary, the LNPssPalmE-Phe(RtoU) is an easy-to-handle drug delivery system (DDS) for delivering mRNA antigens in immunotherapy.

Evaluation of the effects of storage conditions on spray-dried siRNA-LNPs before and after subsequent drying

In an ideal world, pharmaceutical drugs would have infinite shelf life, no susceptibility to degradation, chemical reactions or loss of efficacy. In reality, these processes occur, however, making it desirable to extend a drugs’ shelf life. Nucleic acid-based drugs are most commonly stored as aqueous suspension where they are vulnerable to microbial growth and degradation processes. Drying procedures, such as lyophilization and spray drying, help to reduce the products’ residual moisture while increasing the products’ shelf life and stability. The present study was designed to evaluate 90 days of storage of spray-dried siRNA-lipid nanoparticles (LNPs) at 4 °C and 25 °C. An updated Onpattro® composition modified with a positively charged helper lipid was used as the LNP carrier system. In an attempt to further reduce the residual moisture of our previously reported formulations, all LNP samples were subjected to a secondary drying step in the spray drying tower for 20 min. The measurement of physicochemical properties of spray-dried and subsequently dried LNPs resulted in sizes of 180 nm, PDI values of 0.1–0.15 and zeta potentials of + 3 mV. Spray drying resulted in residual moisture levels of 3.6–4 % and was reduced by subsequent drying to 2.8–3.1 %. Aerodynamic properties after storage showed discrepancies depending on the storage conditions. MMADs remained at 2.8 µm when stored at 4 °C, whereas an increase to 5 µm at 25 °C was observed. Subsequent drying led to sizes of 3.6–3.8 µm, independent of the storage conditions. Spray-dried LNPs maintained bioactivity resulting in > 95 % protein downregulation and confirming the lack of cytotoxic effects in a lung adenocarcinoma cell line. Furthermore, the spray-dried and subsequently dried LNPs stored for 3 months at 4 °C and 25 °C achieved up to 50 % gene silencing of the house-keeping gene GAPDH after deposition on the mucus layer of Calu-3 cells. This study confirms the stability of spray-dried and subsequently dried LNPs over at least 90 days at 4 °C and 25 °C emphasizing the potential of dry powder inhalation of RNA-loaded LNPs as a therapy option for pulmonary diseases.

Nanomechanical action opens endo-lysosomal compartments

Endo-lysosomal escape is a highly inefficient process, which is a bottleneck for intracellular delivery of biologics, including proteins and nucleic acids. Herein, we demonstrate the design of a lipid-based nanoscale molecular machine, which achieves efficient cytosolic transport of biologics by destabilizing endo-lysosomal compartments through nanomechanical action upon light irradiation. We fabricate lipid-based nanoscale molecular machines, which are designed to perform mechanical movement by consuming photons, by co-assembling azobenzene lipidoids with helper lipids. We show that lipid-based nanoscale molecular machines adhere onto the endo-lysosomal membrane after entering cells. We demonstrate that continuous rotation-inversion movement of Azo lipidoids triggered by ultraviolet/visible irradiation results in the destabilization of the membranes, thereby transporting cargoes, such as mRNAs and Cre proteins, to the cytoplasm. We find that the efficiency of cytosolic transport is improved about 2.1-fold, compared to conventional intracellular delivery systems. Finally, we show that lipid-based nanoscale molecular machines are competent for cytosolic transport of tumour antigens into dendritic cells, which induce robust antitumour activity in a melanoma mouse model.

A Workflow for Lipid Nanoparticle (LNP) Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models (SVEM).

We present a Quality by Design (QbD) styled approach for optimizing lipid nanoparticle (LNP) formulations, aiming to offer scientists an accessible workflow. The inherent restriction in these studies, where the molar ratios of ionizable, helper, and PEG lipids must add up to 100%, requires specialized design and analysis methods to accommodate this mixture constraint. Focusing on lipid and process factors that are commonly used in LNP design optimization, we provide steps that avoid many of the difficulties that traditionally arise in the design and analysis of mixture-process experiments by employing space-filling designs and utilizing the recently developed statistical framework of self-validated ensemble models (SVEM). In addition to producing candidate optimal formulations, the workflow also builds graphical summaries of the fitted statistical models that simplify the interpretation of the results. The newly identified candidate formulations are assessed with confirmation runs and optionally can be conducted in the context of a more comprehensive second-phase study.

Exploration of mRNA nanoparticles based on DOTAP through optimization of the helper lipids.

Lipid nanoparticles (LNPs) are one of the most efficient carriers for RNA packaging and delivery, and vaccines based on mRNA-LNPs have received substantial attention since the outbreak of the COVID-19 pandemic. LNPs based on 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) have been widely used in preclinical and clinical settings. A novel non-viral gene delivery system called LNP3 was previously developed, which was composed of DOTAP, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. One of the helper lipids in this carrier was DOPE, which belongs to phospholipids. Given that substituting DOPE with non-phospholipids as helper lipids can increase the delivery efficiency of some LNPs, this study aimed to examine whether non-phospholipids can be formulated with DOTAP as helper lipids. It was found that monoglycerides with C14:0, C16:0, C18:0, C18:1, and C18:2 mediated mRNA transfection, and the transfection efficiency varied between C18:0, C18:1, and C18:2. Furthermore, substituting of the glycerol with other moieties such as the cholesterol or the ethanolamine similarly mediated mRNA transfection. The introduction of cholesterol can further improve the transfection capacity of some DOTAP-based LNPs. One of the best-performing formulations, LNP3-MO, was used to mediate luciferase-mRNA expression in vivo, and the luminescence signal was found to be mainly enriched in the lung and spleen. In addition, the level of SARS-CoV-2 spike antibody in the serum increased after three doses of LNP3-MO mediated SARS-CoV-2 spike mRNA. Altogether, this study demonstrates that non-phospholipids are promising helper lipids that can be formulated with DOTAP to facilitate efficient delivery of mRNAs in vitro and in vivo with organ-specific targeting.

Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

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.

Overview of Solid Lipid Nanoparticles in Breast Cancer Therapy.

Lipid nanoparticles (LNPs), composed of ionized lipids, helper lipids, and cholesterol, provide general therapeutic effects by facilitating intracellular transport and avoiding endosomal compartments. LNP-based drug delivery has great potential for the development of novel gene therapies and effective vaccines. Solid lipid nanoparticles (SLNs) are derived from physiologically acceptable lipid components and remain robust at body temperature, thereby providing high structural stability and biocompatibility. By enhancing drug delivery through blood vessels, SLNs have been used to improve the efficacy of cancer treatments. Breast cancer, the most common malignancy in women, has a declining mortality rate but remains incurable. Recently, as an anticancer drug delivery system, SLNs have been widely used in breast cancer, improving the therapeutic efficacy of drugs. In this review, we discuss the latest advances of SLNs for breast cancer treatment and their potential in clinical use.

Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues

Lipid nanoparticles (LNPs) for delivery of mRNA usually contain ionizable lipid/helper lipid/cholesterol/PEG-lipid in molar ratios of 50:10:38.5:1.5, respectively. These LNPs are rapidly cleared from the circulation following intravenous (i.v.) administration, limiting uptake into other tissues. Here, we investigate the properties of LNP mRNA systems prepared with high levels of "helper" lipids such as 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) or N-(hexadecanoyl)-sphing-4-enine-1-phosphocholine (egg sphingomyelin [ESM]). We show that LNP mRNAs containing 40 mol% DSPC or ESM have a unique morphology with a small interior "solid" core situated in an aqueous compartment that is bounded by a lipid bilayer. The encapsulated mRNA exhibits enhanced stability in the presence of serum. LNP mRNA systems containing 40 mol% DSPC or ESM exhibit significantly improved transfection properties invitro compared with systems containing 10 mol% DSPC or ESM. When injected i.v., LNP mRNAs containing 40 mol% ESM exhibit extended circulation lifetimes compared with LNP mRNA systems containing 10 mol% DSPC, resulting in improved accumulation in extrahepatic tissues. Systems containing 40 mol% ESM result in significantly improved gene expression in spleen and bone marrow as well as liver post i.v. injection compared with 10 mol% DSPC LNP mRNAs. We conclude that LNP mRNAs containing high levels of helper lipid provide a new approach for transfecting hepatic and extrahepatic tissues.

A Combinatorial Library of Lipid Nanoparticles for Cell Type-Specific mRNA Delivery.

Ionizable lipid-based nanoparticles (LNPs) are the most advanced non-viral drug delivery systems for RNA therapeutics and vaccines. However, cell type-specific, extrahepatic mRNA delivery is still a major hurdle, hampering the development of novel therapeutic modalities. Herein, a novel ionizable lipid library is synthesized by modifying hydrophobic tail chains and linkers. Combined with other helper lipids and utilizing a microfluidic mixing approach, stable LNPs are formed. Using Luciferase-mRNA, mCherry mRNA, and Cre mRNA together with a TdTomato animal model, superior lipids forming LNPs for potent cell-type specific mRNA delivery are identified. In vitro assays concluded that combining branched ester tail chains with hydroxylamine linker negatively affects mRNA delivery efficiency. In vivo studies identify Lipid 23 as a liver-trophic, superior mRNA delivery lipid and Lipid 16 as a potent cell type-specific ionizable lipid for the CD11bhi macrophage population without an additional targeting moiety. Finally, in vivo mRNA delivery efficiency and toxicity of these LNPs are compared with SM-102-based LNP (Moderna's LNP formulation) and are shown to be cell-specific compared to SM-102-based LNPs. Overall, this study suggests that a structural combination of tail and linker can drive a novel functionality of LNPs in vivo.

HER2/neu Oncogene Silencing in a Breast Cancer Cell Model Using Cationic Lipid-Based Delivery Systems.

The overexpression of the human epidermal growth factor 2 (HER2/neu) oncogene is predictive of adverse breast cancer prognosis. Silencing the HER2/neu overexpression using siRNA may be an effective treatment strategy. Major requirements for siRNA-based therapy are safe, stable, and efficient delivery systems to channel siRNA into target cells. This study assessed the efficacy of cationic lipid-based systems for the delivery of siRNA. Cationic liposomes were formulated with equimolar ratios of the respective cholesteryl cytofectins, 3β-N-(N', N'-dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) or N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), with the neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE), with and without a polyethylene glycol stabilizer. All cationic liposomes efficiently bound, compacted, and protected the therapeutic siRNA against nuclease degradation. Liposomes and siRNA lipoplexes were spherical, <200 nm in size, with moderate particle size distributions (PDI < 0.4). The siRNA lipoplexes exhibited minimal dose-dependent cytotoxicity and effective HER2/neu siRNA transfection in the HER2/neu overexpressing SKBR-3 cells. The non-PEGylated Chol-T-siRNA lipoplexes induced the highest HER2/neu silencing at the mRNA (10000-fold decrease) and protein levels (>111.6-fold decrease), surpassing that of commercially available Lipofectamine 3000 (4.1-fold reduction in mRNA expression). These cationic liposomes are suitable carriers of HER2/neu siRNA for gene silencing in breast cancer.

Efficient mRNA Delivery with mRNA Lipoplexes Prepared Using a Modified Ethanol Injection Method.

Messenger RNA (mRNA)-based therapies are a novel class of therapeutics used in vaccination and protein replacement therapies for monogenic diseases. Previously, we developed a modified ethanol injection (MEI) method for small interfering RNA (siRNA) transfection, in which cationic liposome/siRNA complexes (siRNA lipoplexes) were prepared by mixing a lipid-ethanol solution with a siRNA solution. In this study, we applied the MEI method to prepare mRNA lipoplexes and evaluated the in vitro and in vivo protein expression efficiencies. We selected six cationic lipids and three neutral helper lipids to generate 18 mRNA lipoplexes. These were composed of cationic lipids, neutral helper lipids, and polyethylene glycol-cholesteryl ether (PEG-Chol). Among them, mRNA lipoplexes containing N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16) or 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl) amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12) with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and PEG-Chol exhibited high protein expression in cells. Furthermore, mRNA lipoplexes composed of DC-1-16, DOPE, and PEG-Chol exhibited high protein expression in the lungs and spleen of mice after systemic injection and induced high antigen-specific IgG1 levels upon immunization. These results suggest that the MEI method can potentially increase the efficiency of mRNA transfection, both in vitro and in vivo.

Development of new non-viral systems for genetic modification of senescent cells

Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population. However, thehigh sensitivity of senescent cells significantly precludes their genetic modification via classical viral and non-viral systems. Niosomes are self-assembled non-viral nanocarriers that exhibit important advantages due to their elevated cytocompatibility, versatility, and cost-efficiency, arising as a new alternative for genetic modification of senescent cells. In this work, we explore for the first time the use of niosomes for genetic modification of senescent umbilical cord-derived mesenchymal stem cells. We report that niosome composition greatly affected transfection efficiency; those formulations prepared in medium with sucrose and containing cholesterol as helper lipid being the most suitable to transfect senescent cells. Moreover, resulting niosome formulations exhibited a superior transfection efficiency with a markedly less cytotoxicity than the commercial reagent Lipofectamine. These findings highlight the potentiality of niosomes as effective vectors for genetic modification of senescent cells, providing new tools for the prevention and/or treatment of age-related diseases.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50–225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM).In contrast, microfluidic mixing resulted in less ordered, notably smaller (50–75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.