Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepared using 300 mm sodium citrate buffer displaying maximum transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.

A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors.

The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.

On the Influence of Nucleic Acid Backbone Modifications on Lipid Nanoparticle Morphology.

Nucleic acid therapeutics represent a major advance toward treating diseases at their root cause. However, nucleic acids are prone to degradation by serum endonucleases, clearance through the immune system, and rapid degradation in complex medium. To overcome these barriers, nucleic acids frequently include chemical modifications to improve stability or decrease immune responses. Lipid nanoparticles (LNPs) have enabled a dramatic reduction in the dose required to achieve a therapeutic effect by protecting these nucleic acids and improving their intracellular delivery. It has been assumed thus far that nonspecific ionic interactions drive LNP formation and chemical modifications to the nucleic acid backbone to confer improved stability do not impact LNP delivery in any way. Here, we demonstrate that these chemical modifications do impact LNP morphology substantially, and phosphorothioate modifications produce stronger interactions with ionizable amino lipids, resulting in enhanced entrapment. This work represents a major first step toward greater understanding of the interaction between the lipid components and nucleic acids within an LNP.

The unusual convergence of steroid catabolic pathways in Mycobacterium abscessus.

Mycobacterium abscessus, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis, Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta-cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaDMab and HsaDMtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (kcat/KM = 1.9 × 104 and 5.7 × 103 mM-1s-1, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaDMab also transformed nonthioesterified substrates efficiently, and a ΔhsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The ΔhsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the ΔhsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

The role of lipid components in lipid nanoparticles for vaccines and gene therapy.

Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases.

Synthesis and Characterization of Hybrid Lipid Nanoparticles Containing Gold Nanoparticles and a Weak Base Drug.

Hybrid lipid nanoparticles containing gold nanoparticles (LNP-GNPs) and drugs have potential for imaging applications as well as triggered release of LNP contents in response to pulsed laser or X-ray radiation mediated by the GNPs. However, methods to synthesize LNP-GNP systems that efficiently entrap GNPs (the potential triggered release and imaging agent) and then load and retain the drug cargo in a manner that may have clinical applications have proven elusive. Here, we develop a straightforward "bottom-up" approach to manufacture drug-loaded LNP-GNP systems. We show that negatively charged GNPs of 5 nm diameter can be stably loaded into LNPs containing 10 mol % ionizable cationic lipid using an ethanol dilution, rapid mixing approach and that these systems also exhibit aqueous compartments. Further, we show that such systems can also entrap ammonium sulfate, enabling pH-dependent loading of the weak base anti-cancer drug doxorubicin into the aqueous compartments. Cryo-transmission electron microscopy (Cryo-TEM) imaging clearly demonstrates the presence of GNPs in the interior of the resulting hybrid nanostructures as well as the formation of electron-dense drug precipitates in the aqueous core of the LNP-GNPs. The approach described here is a robust and straightforward method to generate hybrid LNP-GNP-drug and other LNP-metal nanoparticle-drug systems with potential applications for a variety of triggered release protocols.

Monolignol export by diffusion down a polymerization-induced concentration gradient.

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Modular Lipid Nanoparticle Platform Technology for siRNA and Lipophilic Prodrug Delivery.

Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development.

Scalable Production of Lipid Nanoparticles Containing Amphotericin B.

Lipid-based formulations have been developed to improve stability profiles, tolerability, and toxicity profiles of small molecule drugs. However, manufacture of such formulations involving lipophilic compounds can be labor-intensive and difficult to scale because of solubility and solvent compatibility issues. We have developed a rapid and scalable approach using rapid-mixing techniques to generate homogeneous lipid nanoparticle (LNP) formulations of siRNA, triglycerides, and hydrophilic weak-base drugs. Here, we used this approach to entrap a hydrophobic small molecule, Amphotericin B (AmpB), a hydrophobic drug not soluble in ethanol. The three prototypes presented in this study were derived from LNP-siRNA systems, triglyceride nanoparticles, and liposomal systems. Cryogenic transmission electron microscopy (cryo-TEM) revealed that all three LNP-AmpB formulations retain structural characteristics of the parent (AmpB-free) LNPs, with particles remaining stable for at least 1 month. All formulations showed similar in vitro toxicity profiles in comparison to AmBisome. Importantly, the formulations have a 2.5-fold improved IC50 for fungal growth inhibition as compared to AmBisome in in vitro efficacy studies. These results demonstrate that the rapid-mixing technology combined with dimethyl sulfoxide (DMSO) for drugs insoluble in other organic solvents can be a powerful manufacturing method for the generation of stable LNP drug formulations.

FAM13A as potential therapeutic target in modulating TGF-ß-induced airway tissue remodeling in COPD.

Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-ß1 (TGF-ß1). To determine the role of FAM13A in TGF-ß1 signaling, FAM13A-/- airway epithelial cells were generated using CRISPR-Cas9, whereas overexpression of FAM13A was achieved using lipid nanoparticles. Wild-type (WT) and FAM13A-/- cells were treated with TGF-ß1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGF-ß1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in ß-catenin (CTNNB1) expression in FAM13A-/- cells compared with WT cells after TGF-ß1 treatment. FAM13A overexpression was partially protective from TGF-ß1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared with control nonsmokers, and negatively correlated with lung function. In contrast, ß-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared with non-COPD subjects. Together, our data showed that FAM13A may be protective from TGF-ß1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.

The current landscape of nucleic acid therapeutics.

The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.

mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability.

A drawback of the current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at (ultra)low temperatures. Understanding the root cause of the instability of these vaccines may help to rationally improve mRNA-LNP product stability and thereby ease the temperature conditions for storage. In this review we discuss proposed structures of mRNA-LNPs, factors that impact mRNA-LNP stability and strategies to optimize mRNA-LNP product stability. Analysis of mRNA-LNP structures reveals that mRNA, the ionizable cationic lipid and water are present in the LNP core. The neutral helper lipids are mainly positioned in the outer, encapsulating, wall. mRNA hydrolysis is the determining factor for mRNA-LNP instability. It is currently unclear how water in the LNP core interacts with the mRNA and to what extent the degradation prone sites of mRNA are protected through a coat of ionizable cationic lipids. To improve the stability of mRNA-LNP vaccines, optimization of the mRNA nucleotide composition should be prioritized. Secondly, a better understanding of the milieu the mRNA is exposed to in the core of LNPs may help to rationalize adjustments to the LNP structure to preserve mRNA integrity. Moreover, drying techniques, such as lyophilization, are promising options still to be explored.

Altering the intra-liver distribution of phospholipid-free small unilamellar vesicles using temperature-dependent size-tunability.

We demonstrated that phospholipid-free small unilamellar vesicles (PFSUVs) composed of TWEEN 80 and cholesterol (25/75, mol%) could be fabricated using a staggered herringbone micromixer with precise controlling of their mean size between 54 nm and 147 nm. Increasing the temperature or decreasing the flow rate led to an increase in the resulting particle diameter. In zebrafish embryos, 120-nm PFSUVs showed 3-fold higher macrophage clearance compared to the 60-nm particles, which exhibited prolonged blood circulation. In mice, the 60-nm particles showed dominant accumulation in the liver hepatocytes (66% hepatocytes positive), while the 120-nm particles were delivered equally to the liver and spleen macrophages. Accordingly, in a murine model of acetaminophen-induced hepatotoxicity the 60-nm particles loaded with chlorpromazine reduced the serum alanine aminotransferase level and liver necrosis 2- to 4-fold more efficiently than their 120-nm counterparts and the free drug, respectively. This work showed that the intra-liver distribution of PFSUVs was largely determined by the size. Most other nanoparticles published to date are predominantly cleared by the liver Kupffer cells. The 60-nm PFSUVs, on the other hand, focused the delivery to the hepatocytes with significant advantages for the therapy of liver diseases.

Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations.

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Characterization of Lipid Nanoparticles Containing Ionizable Cationic Lipids Using Design-of-Experiments Approach.

Lipid nanoparticles (LNPs) containing short-interfering RNA (LNP-siRNA systems) are a promising approach for silencing disease-causing genes in hepatocytes following intravenous administration. LNP-siRNA systems are generated by rapid mixing of lipids in ethanol with siRNA in aqueous buffer (pH 4.0) where the ionizable lipid is positively charged, followed by dialysis to remove ethanol and to raise the pH to 7.4. Ionizable cationic lipids are the critical excipient in LNP systems as they drive entrapment and intracellular delivery. A recent study on the formation of LNP-siRNA systems suggested that ionizable cationic lipids segregate from other lipid components upon charge neutralization to form an amorphous oil droplet in the core of LNPs. This leads to a decrease in intervesicle electrostatic repulsion, thereby engendering fusion of small vesicles to form final LNPs of increased size. In this study, we prepared LNP-siRNA systems containing four lipid components (hydrogenated soy phosphatidylcholine, cholesterol, PEG-lipid, and 1,2-dioleoyl-3-dimethylammonium propane) by microfluidic mixing. The effects of preparation parameters [lipid concentration, flow rate ratio (FRR), and total flow rate], dialysis process, and complex formation between siRNA and ionizable cationic lipids on the physicochemical properties [siRNA entrapment on the particle size and polydispersity index (PDI)] were investigated using a design of experiments approach. The results for the preparation parameters showed no impact on siRNA encapsulation, but lipid concentration and FRR significantly affected the particle size and PDI. In addition, the effect of FRR on the particle size was suppressed in the presence of anionic polymers such as siRNA as compared to the case of LNPs alone. More intriguingly, unlike empty LNPs, a decrease in the PDI and an increase in the particle size occurred after dialysis in the LNP-siRNA systems. Such changes by dialysis were suppressed at FRR = 1. These findings provide useful information to guide the development and manufacturing conditions for LNP-siRNA systems.