Charged Lipids Research Articles

Charge-Stabilized Nanodiscs as a New Class of Lipid Nanoparticles.

Nanoparticles have the potential to improve disease treatment and diagnosis due to their ability to incorporate drugs, alter pharmacokinetics, and enable tissue targeting. While considerable effort is placed on developing spherical lipid-based nanocarriers, recent evidence suggests that high aspect ratio lipid nanocarriers can exhibit enhanced disease site targeting and altered cellular interactions. However, the assembly of lipid-based nanoparticles into non-spherical morphologies has typically required incorporating additional agents such as synthetic polymers, proteins, lipid-polymer conjugates, or detergents. Here, charged lipid headgroups are used to generate stable discoidal lipid nanoparticles from mixed micelles, which are termed charge-stabilized nanodiscs (CNDs). The ability to generate CNDs in buffers with physiological ionic strength is restricted to lipids with more than one anionic group, whereas monovalent lipids only generate small nanoliposomal assemblies. In mice, the smaller size and anisotropic shape of CNDs promote higher accumulation in subcutaneous tumors than spherical liposomes. Further, the surface chemistry of CNDs can be modified via layer-by-layer (LbL) assembly to improve their tumor-targeting properties over state-of-the-art LbL-liposomes when tested using a metastatic model of ovarian cancer. The application of charge-mediated anisotropy in lipid-based assemblies can aid in the future design of biomaterials and cell-membrane mimetic structures.

Functional interplay between short antimicrobial peptides and model lipid membranes

Antimicrobial peptides (AMPs) are considered an attractive generation of novel antibiotics due to their advantageous properties such as a broad spectrum of antimicrobial activity against pathogens, low cytotoxicity, and drug resistance. Although they have common structural features and it has been widely demonstrated that bacterial membranes represent the main target of the peptide activity, the exact mechanism underlying the membrane perturbation by AMPs is not fully understood. Nevertheless, all the proposed modes of action implicate the preliminary interaction of AMPs with the negatively charged lipids in bacterial membranes. Recently, the structural and functional characterization of two AMPs, RiLK1 and RiLK3, was reported. Specifically, both peptides were revealed to be multitalented compounds capable of binding Gram-positive and Gram-negative liposome models with high affinity, but their mechanism of action remains elusive. In this paper, the effects of RiLK1 and RiLK3 on vesicles mimicking prokaryotic and eukaryotic cell membranes were further examined by using different approaches. Fluorescence and quenching assays either by acrylamide or lipophilic probes suggested that the peptides were mainly located at the interface of the negatively charged membranes that mimicked those of Salmonella Typhimurium and Staphylococcus aureus, possibly oriented in a parallel manner. Furthermore, RiLK1 and RiLK3 caused a significant leakage of carboxyfluorescein from bacterial liposomes, demonstrating that they can permeabilize the target membranes at high doses. Conversely, both peptides appear to behave like cell penetrating peptides (CPPs) at concentrations near their MIC values evaluated against the bacterial targets. Moreover, Dynamic Light Scattering provided further insights on the mechanisms of antimicrobial peptide against the bacterial liposomes. Conclusively, in vitro experiments indicated that RiLK1 and RiLK3 displayed potent bacteriostatic efficacy at low micromolar concentrations against an antibiotic-resistant ESKAPE pathogen, making them a valuable tool in preventing and treating infections caused by such bacteria.

Implications of the Lipidic Ecosystem for the Membrane Binding of ApoE Signal Peptide: Importance of Sphingomyelin.

The unidirectional movement of nascent secretory proteins in the cell is primarily assisted by the signal recognition particles (SRP). However, this does not completely justify the importance of the signal peptide (SP) which gets eliminated after the protein translocation. We have earlier demonstrated that a negatively charged lipid such as POPG plays an important role in the higher binding affinity and cholesterol-discriminating ability of the apolipoprotein E (ApoE) SP. In this present work, we aimed to understand the role of sphingomyelin, an important constituent of ER, on the membrane binding of ApoE SP. Our results demonstrate that sphingomyelin promotes membrane binding but cannot discriminate cholesterol. However, sphingomyelin shows a synergistic effect with POPG toward the membrane binding of the ApoE SP. We have further shown that the membrane domains do not have any impact on the binding of ApoE SP. Based on our results we propose that the lipid composition of the endoplasmic reticulum (ER) where ApoE translocates, enhances the binding of the ApoE signal peptide to the ER membrane.

Factors Promoting Lipopolysaccharide Uptake by Synthetic Lipid Droplets.

Lipoproteins are essential in removing lipopolysaccharide (LPS) from blood during bacterial inflammation. The physicochemical properties of lipoproteins and environmental factors can impact LPS uptake. In this work, synthetic lipid droplets containing triglycerides, cholesterols, and phospholipids, were prepared to mimic lipoproteins. The physicochemical properties of these lipid droplets, such as charges, sizes, and lipid compositions, were altered to understand the underlying factors affecting LPS uptake. The amphiphilic LPS could spontaneously adsorb on the surface of lipid droplets without lipopolysaccharide binding protein (LBP); however, the presence of LBP can increase LPS uptake. The positively charged lipid droplets also enhance the uptake of negatively charged LPS. Most interestingly, the LPS uptake highly depends on the concentrations of Ca 2+ near the physiological conditions, but the impact of Mg 2+ ions was not significant. The increase of Ca 2+ ions can improve LPS uptake by lipid droplets; this result suggested that Ca 2+ may play an essential role in LPS clearance. Since septic shock patients typically suffer from hypocalcemia and low levels of lipoproteins, the supplementation of Ca 2+ ions along with synthetic lipoproteins may be a potential treatment for severe sepsis.

Membrane Composition Allows the Optimization of Berberine Encapsulation in Liposomes.

Berberine (BBR) is a natural molecule with noteworthy pharmacological properties, including the prevention of antibiotic resistance in Gram-negative bacteria. However, its oral bioavailability is poor, thus resulting in an impaired absorption and efficacy in humans. In combination with other drugs, liposomes have been shown to enhance the availability of the drug, representing a smart delivery system to target tissues and reduce negative side effects. To date, there is a lack of studies on BBR and liposomes that enable the rationalization and molecular-based design of such formulations for future use in humans. In this work, the encapsulation of BBR into liposomes is proposed to overcome current limitations using a combination of experimental and computational assays to rationalize the membrane composition of liposomes that maximizes BBR encapsulation. First, the encapsulation efficiency was measured for several membrane compositions, revealing that it is enhanced by cholesteryl hemisuccinate and, to a lesser extent, by cholesterol. The physical basis of the BBR encapsulation efficiency and permeability was clarified using molecular dynamics simulation: using the lipid composition, one can tune the capability of membranes to attract, i.e., to adsorb, the molecules onto their surface. Overall, these findings suggest a rational strategy to maximize the encapsulation efficiency of liposomes by using negatively charged lipids, thus representing the basis for designing delivery systems for BBR, useful to treat, e.g., antibiotic resistance.

Electroformation of Giant Unilamellar Vesicles from Damp Films in Conditions Involving High Cholesterol Contents, Charged Lipids, and Saline Solutions.

Giant unilamellar vesicles (GUVs) are frequently used as membrane models in studies of membrane properties. They are most often produced using the electroformation method. However, there are a number of parameters that can influence the success of the procedure. Some of the most common conditions that have been shown to have a negative effect on GUV electroformation are the presence of high cholesterol (Chol) concentrations, the use of mixtures containing charged lipids, and the solutions with an elevated ionic strength. High Chol concentrations are problematic for the traditional electroformation protocol as it involves the formation of a dry lipid film by complete evaporation of the organic solvent from the lipid mixture. During drying, anhydrous Chol crystals form. They are not involved in the formation of the lipid bilayer, resulting in a lower Chol concentration in the vesicle bilayer compared to the original lipid mixture. Motivated primarily by the issue of artifactual Chol demixing, we have modified the electroformation protocol by incorporating the techniques of rapid solvent exchange (RSE), ultrasonication, plasma cleaning, and spin-coating for reproducible production of GUVs from damp lipid films. Aside from decreasing Chol demixing, we have shown that the method can also be used to produce GUVs from lipid mixtures with charged lipids and in ionic solutions used as internal solutions. A high yield of GUVs was obtained for Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) samples with mixing ratios ranging from 0 to 2.5. We also succeeded in preparing GUVs from mixtures containing up to 60 mol% of the charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and in NaCl solutions with low ionic strength (<25 mM).

Characterization of a Charged Biomimetic Lipid Membrane for Unique Antifouling Effects against Clinically Relevant Matrices in Biosensing.

Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Machine Learning Elucidates Design Features of Plasmid Deoxyribonucleic Acid Lipid Nanoparticles for Cell Type-Preferential Transfection.

To broaden the accessibility of cell and gene therapies, it is essential to develop and optimize nonviral, cell type-preferential gene carriers such as lipid nanoparticles (LNPs). While high-throughput screening (HTS) approaches have proven effective in accelerating LNP discovery, they are often costly, labor-intensive, and do not consistently yield actionable design rules that direct screening efforts toward the most relevant chemical and formulation parameters. In this study, we employed a machine learning (ML) workflow, utilizing well-curated plasmid DNA LNP transfection data sets across six cell types, to extract compositional and chemical insights from HTS studies. Our approach achieved prediction errors averaging between 5 and 10%, depending on the cell type. By applying SHapley Additive exPlanations to our ML models, we uncovered key composition-function relationships that govern cell type-preferential LNP transfection efficiency. Notably, we identified consistent LNP composition parameters that enhance in vitro transfection efficiency across diverse cell types, including a helper lipid molar percentage of charged lipids between 9 and 50% and the inclusion of cationic/zwitterionic helper lipids. Additionally, several parameters were found to modulate cell type-preferentiality, such as the total molar percentage of ionizable and helper lipids, N/P ratio, PEGylated lipid molar percentage of uncharged lipids, and hydrophobicity of the helper lipid. This study leverages HTS of compositionally diverse LNP libraries combined with ML analysis to elucidate the interactions between lipid components in LNP formulations, providing insights that contribute to the design of LNP compositions tailored for cell type-preferential transfection.

An Extensive Review on Novel Liposomes : Classification, Methodology, Characterization, Current Formulations.

Liposomes are vesicles consisting of a phospholipid, hydrophobic drug, hydrophobic tail, hydrophilic tail, cholesterol, targeting agents, positive and negatively charged lipids, and a drug encapsulated in the center of the phospholipid group having a spherical shape. The phospholipid consists of an equal number of aqueous membranes, making the liposomes an important nanocarrier for the drug delivery to the targeted site. Various liposome-based products have recently been approved and are in clinical trials. This review will discuss the structure, classification, types, and method of liposome preparation and various marketed liposomal products.

Characterization of NK-lysin A, a potent antimicrobial peptide from the zebrafish Danio rerio

Antimicrobial peptides (AMPs) are important players of the innate immune system with a major role in the defense against invading pathogens. AMPs belonging to the family of saposin-like proteins (SAPLIPs) include the porcine NK-lysin and the human granulysin. In the zebrafish Danio rerio, transcript analyses of NK-lysin encoding genes have been reported, but biochemical characterizations at the protein level are missing so far. Here, we present the recombinant expression, purification, and characterization of one of these homologs, namely of NK-lysin A (DaNKlA). To remove the affinity tag from DaNKlA, we made use of a self-splicing intein. Recombinant DaNKlA depolarized liposomes over a broad pH range and showed a preference for negatively charged lipids. DaNKlA inhibited the growth of and killed different Gram-positive and Gram-negative bacteria, including the fish pathogenic bacterium Vibrio anguillarum, by membrane permeabilization but displayed substantially lower activity against yeast cells. Structural modelling and bioinformatic comparison of DaNKlA with characterized SAPLIPs suggest membrane destabilization accompanied by strong electrostatic interactions as the mode of action.

S-acylation of NLRP3 provides a nigericin sensitive gating mechanism that controls access to the Golgi.

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

Turning a negative into a positive.

Negatively charged lipid bilayers enhance the interaction between a chemokine and an atypical chemokine receptor.

Secondary structure propensities of the Ebola delta peptide E40 in solution and model membrane environments

The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na+ ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles.

Discovery of a Peptoid-Based Nanoparticle Platform for Therapeutic mRNA Delivery via Diverse Library Clustering and Structural Parametrization.

Nanoparticle-mediated mRNA delivery has emerged as a promising therapeutic modality, but its growth is still limited by the discovery and optimization of effective and well-tolerated delivery strategies. Lipid nanoparticles containing charged or ionizable lipids are an emerging standard for in vivo mRNA delivery, so creating facile, tunable strategies to synthesize these key lipid-like molecules is essential to advance the field. Here, we generate a library of N-substituted glycine oligomers, peptoids, and undertake a multistage down-selection process to identify lead candidate peptoids as the ionizable component in our Nutshell nanoparticle platform. First, we identify a promising peptoid structural motif by clustering a library of >200 molecules based on predicted physical properties and evaluate members of each cluster for reporter gene expression in vivo. Then, the lead peptoid motif is optimized using design of experiments methodology to explore variations on the charged and lipophilic portions of the peptoid, facilitating the discovery of trends between structural elements and nanoparticle properties. We further demonstrate that peptoid-based Nutshells leads to expression of therapeutically relevant levels of an anti-respiratory syncytial virus antibody in mice with minimal tolerability concerns or induced immune responses compared to benchmark ionizable lipid, DLin-MC3-DMA. Through this work, we present peptoid-based nanoparticles as a tunable delivery platform that can be optimized toward a range of therapeutic programs.

Resolving the interactions between hydrophilic CdTe quantum dots and positively charged membranes at the nanoscale

The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.

FAIMS Shotgun Lipidomics for Enhanced Class- and Charge-State Separation Complemented by Automated Ganglioside Annotation.

The analysis of gangliosides is extremely challenging, given their structural complexity, lack of reference standards, databases, and software solutions. Here, we introduce a fast 6 min high field asymmetric ion mobility spectrometry (FAIMS) shotgun lipidomics workflow, along with a dedicated software solution for ganglioside detection. By ramping FAIMS compensation voltages, ideal ranges for different ganglioside classes were obtained. FAIMS revealed both class- and charge-state separation behavior based on the glycan headgroup moiety. The number of sialic acids attached to the glycan moiety correlates positively with their preferred charge states, i.e., trisialylated gangliosides were mainly present as [M - 3H]3- ions, whereas [M - 4H]4- and [M - 5H]5- ions were observed for GQ1 and GP1. For data evaluation, we developed a shotgun/FAIMS extension for the open-source Lipid Data Analyzer (LDA), enabling automated annotation of gangliosides up to the molecular lipid species level. This extension utilized combined orthogonal fragmentation spectra from CID, HCD, and 213 nm UVPD ion activation methods and covers 29 ganglioside classes, including acetylated and fucosylated modifications. With our new workflow and software extension 117 unique gangliosides species were identified in porcine brain extracts. While conventional shotgun lipidomics favored the observation of singly charged ganglioside species, the utilization of FAIMS made multiply charged lipid species accessible, resulting in an increased number of detected species, primarily due to an improved signal-to-noise ratio arising from FAIMS charge state filtering. Therefore, this FAIMS-driven workflow, complemented by new software capabilities, offers a promising strategy for complex ganglioside and glycosphingolipid characterization in shotgun lipidomics.

Interplay between Copper, Phosphatidylserine, and α-Synuclein Suggests a Link between Copper Homeostasis and Synaptic Vesicle Cycling.

Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.

Differential effects of ganglioside lipids on the conformation and aggregation of islet amyloid polypeptide.

Despite causing over 1 million deaths annually, Type 2 Diabetes (T2D) currently has no curative treatments. Aggregation of the islet amyloid polypeptide (hIAPP) into amyloid plaques plays an important role in the pathophysiology of T2D and thus presents a target for therapeutic intervention. The mechanism by which hIAPP aggregates contribute to the development of T2D is unclear, but it is proposed to involve disruption of cellular membranes. However, nearly all research on hIAPP-lipid interactions has focused on anionic phospholipids, which are primarily present in the cytosolic face of plasma membranes. We seek here to characterize the effects of three gangliosides, the dominant anionic lipids in the outer leaflet of the plasma membrane, on the aggregation, structure, and toxicity of hIAPP. Our results show a dual behavior that depends on the molar ratio between the gangliosides and hIAPP. For each ganglioside, a low-lipid:peptide ratio enhances hIAPP aggregation and alters the morphology of hIAPP fibrils, while a high ratio eliminates aggregation and stabilizes an α-helix-rich hIAPP conformation. A more negative lipid charge more efficiently promotes aggregation, and a larger lipid headgroup improves inhibition of aggregation. hIAPP also alters the phase transitions of the lipids, favoring spherical micelles over larger tubular micelles. We discuss our results in the context of the available lipid surface area for hIAPP binding and speculate on a role for gangliosides in facilitating toxic hIAPP aggregation.

Synaptotagmin-1 undergoes phase separation to regulate its calcium-sensitive oligomerization.

Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.

The 60-year evolution oflipid nanoparticles fornucleic acid delivery.

Delivery of genetic information to the interior of target cells in vivo has been a major challenge facing gene therapies. This barrier is now being overcome, owing in part to dramatic advances made by lipid-based systems that have led to lipid nanoparticles (LNPs) that enable delivery ofnucleic acid-based vaccines and therapeutics. Examples include the clinically approved COVID-19 LNP mRNA vaccines and Onpattro (patisiran), an LNP small interfering RNA therapeutic to treat transthyretin-induced amyloidosis (hATTR). In addition, a host of promising LNP-enabled vaccines and gene therapies are in clinical development. Here, we trace this success to two streams of research conducted over the past 60 years: the discovery of the transfection properties of lipoplexes composed of positively charged cationic lipids complexed with nucleic acid cargos and the development of lipid nanoparticles using ionizable cationic lipids. The fundamental insights gained from these two streams of research offer potential delivery solutions for most forms of gene therapies.