Lipid Nanoparticle Formulations Research Articles

Lipid nanoparticles deliver DNA-encoded biologics and induce potent protective immunity

Lipid nanoparticles (LNPs) for mRNA delivery have advanced significantly, but LNP-mediated DNA delivery still faces clinical challenges. This study compared various LNP formulations for delivering DNA-encoded biologics, assessing their expression efficacy and the protective immunity generated by LNP-encapsulated DNA in different models. The LNP formulation used in Moderna’s Spikevax mRNA vaccine (LNP-M) demonstrated a stable nanoparticle structure, high expression efficiency, and low toxicity. Notably, a DNA vaccine encoding the spike protein, delivered via LNP-M, induced stronger antigen-specific antibody and T cell immune responses compared to electroporation. Single-cell RNA sequencing (scRNA-seq) analysis revealed that the LNP-M/pSpike vaccine enhanced CD80 activation signaling in CD8+ T cells, NK cells, macrophages, and DCs, while reducing the immunosuppressive signals. The enrichment of TCR and BCR by LNP-M/pSpike suggested an increase in immune response specificity and diversity. Additionally, LNP-M effectively delivered DNA-encoded antigens, such as mouse PD-L1 and p53R172H, or monoclonal antibodies targeting mouse PD1 and human p53R282W. This approach inhibited tumor growth or metastasis in several mouse models. The long-term anti-tumor effects of LNP-M-delivered anti-p53R282W antibody relied on memory CD8+ T cell responses and enhanced MHC-I signaling from APCs to CD8+ T cells. These results highlight LNP-M as a promising and effective platform for delivering DNA-based vaccines and cancer immunotherapies.

Ionizable Cholesterol Analogs as the Fifth Component of Lipid Nanoparticles for Selective Targeting Delivery of mRNA.

Lipid nanoparticles (LNP) have shown great promise in clinical applications for delivering mRNA. Targeted delivery of mRNA to particular tissues or organs is essential for precise therapeutic outcomes and minimized side effects in various disease models. However, achieving targeted delivery beyond the liver is a challenge based on current LNP formulations. In this report, we synthesized four ionizable cholesterol analogs by attaching two tertiary amine groups onto the head of a cholesterol-like structure and incorporated them as a fifth component into conventional commercial LNPs based on ALC-0315 or SM-102. Selective targeting delivery of mRNA is achieved by adjusting the proportion of the fifth component in the LNP. Specifically, a spleen-targeted 0315-Ergo-40% formulation demonstrated an impressive 95% delivery efficiency, while a lung-targeted 102-Sito-40% formulation achieved up to 78%. Moreover, when this strategy is applied to a self-developed ionizable lipid named U-101 instead of ALC-0315 or SM-102, the targeting delivery efficiencies to the spleen and lungs reach 96 and 71%, respectively. Multiple assessments suggest that inclusion of the fifth component does not compromise LNP stability, as indicated by consistent particle size, polydispersity index (PDI), and encapsulation efficiency. Furthermore, the test results of liver and kidney function and immunogenicity reveal no increase of toxicity in vivo following the introduction of the fifth component. Additional studies on in vitro cytotoxicity, lysosomal escape, and cellular transfection efficiency confirm that the fifth component does not diminish delivery performance. Taken together, the incorporation of ionizable cholesterol analogs leads to targeting of LNP delivery, which features strong organ selectivity, high safety, and suitability for further evaluation.

Optimization of Docetaxel-Zedoary Turmeric Oil Magnetic Solid Lipid Nanoparticle Preparation by Central Composite Design-Response Surface Methodology.

To optimize the formulation of docetaxel-zedoary oil magnetic solid lipid nanoparticles (DTX-ZTO-MSLN) using central composite design-response surface methodology. First, the formulation and preparation process of DTX-ZTO-MSLN were optimized via design-response surface methodology. The appearance, particle size, thermogravimetric, pH, iron content, magnetic strength, and in vitro drug release of DTX-ZTO-MSLN were subsequently examined. Finally, the antitumor effect of DTX-ZTO-MSLN on MCF-7 breast cancer cells was measured via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The optimized formulation was as follows: the mass ratio of soybean phospholipid to poloxamer 188 was 0.34, the mass ratio of DTX-ZTO to glycerol monostearate was 3.23, and 29.42 mL of water was used. The DTX-ZTO-MSLN prepared by the optimized method was clear and transparent, with good stability, with an iron content of 7.38%, and a saturation magnetization intensity of 7.05 A·m2·kg-1. The in vitro drug release was consistent with the Weibull model (R2 = 0.9992). Compared with zedoary turmeric oil and docetaxel, DTX-ZTO-MSLN had a much greater inhibitory effect on MCF-7 cells (p < 0.05). The optimized DTX-ZTO-MSLN meets the quality requirements for nanoemulsions. This study provides a theoretical basis for developing and applying DTX-ZTO-MSLN.

Multiarm-Assisted Design of Dendron-like Degradable Ionizable Lipids Facilitates Systemic mRNA Delivery to the Spleen.

Lipid nanoparticles (LNPs) have emerged as pivotal vehicles for messenger RNA (mRNA) delivery to hepatocytes upon systemic administration and to antigen-presenting cells following intramuscular injection. However, achieving systemic mRNA delivery to non-hepatocytes remains challenging without the incorporation of targeting ligands such as antibodies, peptides, or small molecules. Inspired by comb-like polymeric architecture, here we utilized a multiarm-assisted design to construct a library of 270 dendron-like degradable ionizable lipids by altering the structures of amine heads and multiarmed tails for optimal mRNA delivery. Following in vitro high-throughput screening, a series of top-dendron-like LNPs with high transfection efficacy were identified. These dendron-like ionizable lipids facilitated greater mRNA delivery to the spleen in vivo compared to ionizable lipid analogs lacking dendron-like structure. Proteomic analysis of corona-LNP pellets showed enhancement of key protein clusters, suggesting potential endogenous targeting to the spleen. A lead dendron-like LNP formulation, 18-2-9b2, was further used to encapsulate Cre mRNA and demonstrated excellent genome modification in splenic macrophages, outperforming a spleen-tropic MC3/18PA LNP in the Ai14 mice model. Moreover, 18-2-9b2 LNP encapsulating therapeutic BTB domain and CNC homologue 1 (BACH1) mRNA exhibited proficient BACH1 expression and subsequent Spic downregulation in splenic red pulp macrophages (RPM) in a Spic-GFP transgene model upon intravenous administration. These results underscore the potential of dendron-like LNPs to facilitatem RNA delivery to splenic macrophages, potentially opening avenues for a range of mRNA-LNP therapeutic applications, including regenerative medicine, protein replacement, and gene editing therapies.

Formulation, Characterization, and Cytotoxic Effect of Indomethacin-Loaded Nanoparticles.

Indomethacin (IND), classified as class 2 in the Biopharmaceutical Classification System (BCS), has emerged as an anti-inflammatory agent with low solubility and high permeability. Widely used in the treatment of various diseases, such as rheumatoid arthritis and ankylosing spondylitis, this drug is well-known for its adverse effects, particularly in the stomach, and a short biological half-life, which is around 1.5-2 hours. The aim of this study was to overcome the challenges of low solubility, short half-life, and serious side effects occurring with the use of IND-loaded formulations of Solid Lipid Nanoparticles (SLNs) and Polymeric Nanoparticles (PNPs). For PNPs, emulsification/solvent evoporation method was employed, and for SLNs, the hot homogenizaton method was applied. Eudragit® RLPO (RLPO) and Eudragit® RSPO (RSPO) were used as polymers for PNP and Dynasan®116 (DYN) was used as the solid lipid for SLN. Prepared formulations were characterized for Particle Size (PS), Poly-dispersity Index (PDI), Zeta Potential (ZP), Encapsulation Efficiency (%EE), and drug-ex-recipient compatibility using DSC, FT-IR, and 1H NMR; cumulative drug release rates were assessed using HPLC and in vitro cytotoxicities were examined by the MTT assay. Both PNP and SLN formulations' zeta potential, particle size, and PDI results indicated the formulations to have good stability. Encapsulation efficiency values were obtained as desired. Drug-excipient compatibility was proved using DSC, FT-IR, and 1H NMR. In vitro dissolution results have proven both formulations to have longer release than pure indomethacin. In the MTT analysis of indomethacin application for 24 and 48 hours, a linear correlation was observed between drug concentration and cell viability, and it was determined that the PNP formulation exhibited fewer toxic effects among the formulations. This has proven the PNP nanocarrier as safer for normal cells. IND-loaded PNP and SLN formulations have been successfully prepared in this work and they have achieved drug release in the intestine and prolonged the release duration.

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery.

Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use. These characteristics have been improved through the development of microfluidic platforms, which have enhanced the efficacy and uniformity of LNP batches. However, our understanding of how the composition and mixing method influences the structural, biophysical, and biological properties of the resulting particles remains limited, hindering the development of LNPs. Our lack of structural understanding extends from the physical and compositional polydispersity of LNPs, which render traditional characterization methods, such as dynamic light scattering (DLS), unable to accurately quantitate the physicochemical characteristics of LNPs. In this study, we address the challenge of structurally characterizing polydisperse LNP formulations using emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation (SV-AUC), field flow fractionation followed by multi-angle light scattering (FFF-MALS), and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering (SEC-SAXS). Here, we show that the LNPs have intrinsic polydispersity in size, RNA loading, and shape, and that these parameters are dependent on both the formulation technique and lipid composition. Lastly, we demonstrate that these biophysical methods can be employed to predict transfection in three biological models by examining the relationship between mRNA translation and physicochemical characteristics. We envision that employing solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for future LNPs.

Placenta-tropic VEGF mRNA lipid nanoparticles ameliorate murine pre-eclampsia.

Pre-eclampsia is a placental disorder that affects 3-5% of all pregnancies and is a leading cause of maternal and fetal morbidity worldwide1,2. With no drug available to slow disease progression, engineering ionizable lipid nanoparticles (LNPs) for extrahepatic messenger RNA (mRNA) delivery to the placenta is an attractive therapeutic option for pre-eclampsia. Here we use high-throughput screening to evaluate a library of 98 LNP formulations in vivo and identify a placenta-tropic LNP (LNP 55) that mediates more than 100-fold greater mRNA delivery to the placenta in pregnant mice than a formulation based on theFood and Drug Administration-approved Onpattro LNP (DLin-MC3-DMA)3. We propose an endogenous targeting mechanism based on β2-glycoprotein I adsorption that enables LNP delivery to the placenta. In both inflammation- and hypoxia-induced models of pre-eclampsia, a single administration of LNP 55 encapsulating vascular endothelial growth factor (VEGF) mRNA resolves maternal hypertensionuntil the end of gestation. In addition, with our VEGF mRNA LNP 55 therapeutic, we demonstrate improvements in fetal health and partially restore placental vasculature, the local and systemic immune landscape and serum levels of soluble Fms-like tyrosine kinase-1, a clinical biomarker of pre-eclampsia1. Together, these results demonstrate the potential of this mRNA LNP platform for treating placental disorders such as pre-eclampsia.

Model Adequacy in Assessing the Predictive Performance of Regression Models in Pharmaceutical Product Optimization: The Bedaquiline Solid Lipid Nanoparticle Example

This study aimed to assess the predictive performance of first- and second-order regression models in optimizing bedaquiline (BQ) solid lipid nanoparticle (SLN) formulations. A three-step central composite design and graphical optimization process was employed. A design of experiments method was used to evaluate the impact of BQ, Tween 80 (T80), polyethylene glycol (PEG), and lecithin on the formulations’ response variables, including Z-average (PSD), polydispersibility index (PdI), and Zeta potential (ZP). Secondly, we quantified the relationship between experimental variables using the regression model coefficients. Lastly, we predicted the responses and verified the models’ adequacies to ensure accurate representation and effective optimization. The first-order polynomial showed poor model adequacy and required further refinement due to its lack of explanatory power and significant predictors. Conversely, the second-order models provided superior fitness, sensitivity to variability, complexity, and prediction consistency. The optimized formulation achieved a desirability value of 0.9998, indicating alignment with the desired criteria. Specifically, the levels of BQ (19.4 mg), T80 (25.2 mg), PEG (39.2 mg), and lecithin (200 mg) corresponded to PdI (0.41), PSD (250.99 nm), and ZP (−25.95 mV). Maintaining a BQ concentration between 10 and 20% and T80 between 15 and 18% is vital for maximizing ZP and minimizing PdI and PSD, ensuring stable SLN formulations. This study underscores the significance of precise model selection and statistical analysis in pharmaceutical formulation optimization for enhanced drug delivery systems.

Formulation of Solid Lipid Nanoparticles Loaded with Rosiglitazone and Probiotic: Optimization and In-vitro Characterization.

In the present study, solid lipid nanoparticles loaded with Rosiglitazone and probiotics were prepared via solvent emulsification diffusion method which is patented. As a lipid and surfactant, Gleceryl monostearate and Pluronic -68 were used in the formulation process. During characterization, it was determined that ingredient quantity variations significantly impacted Rosiglitazone loading capacity, particle size, polydispersity index, etc. In an optimized formulation of RSG-PB loaded SLNs, spherical particles with a mean particle size of 147.66 ± 1.52 nm, PDI of 0.42 ± 0.02, and loading capacity of 45.36 ± 0.20 were identified. Moreover, the developed SLNs had the potential to discharge the drug for up to 24 hours, as predicted by Higuchi's pharmacokinetic model. The SLNs were stable at 25°C/60%RH for up to 60 days. There was little to no change in particle size, PDI, or loading capacity. In addition, the number of probiotic bacteria was determined using the standard plate count procedure. Further, the antioxidant effect of the prepared formulation is evaluated using the DPPH assay method. This study concludes that the method used to fabricate RSG-probiotic-loaded SLNs is straightforward and yields favorable results regarding various parameters, including sustained release property, particle size, PDI, and percent drug loading stability. Furthermore, DPPH radical scavenging activity shows the high antioxidant potential of RSG-PB SLNs when compared to RSG and probiotics alone.

Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.

Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.

Lipid Nanoparticles With Fine-Tuned Composition Show Enhanced Colon Targeting as a Platform for mRNA Therapeutics.

Lipid Nanoparticles (LNPs) recently emerged as an invaluable RNA delivery platform. With many LNP-based therapeutics in the pre-clinical and clinical pipelines, there is extensive research dedicated to improving LNPs. These efforts focus mainly on the tolerability and transfectability of new ionizable lipids and RNAs, or modulating LNPs biodistribution with active targeting strategies. However, most formulations follow the well-established lipid proportions used in clinically approved products. Nevertheless, investigating the effects of LNPs composition on their biodistribution can expand the toolbox for particle design, leading to improved delivery strategies. Herein, a new LNPs (30-n-LNPs) formulation with increasing amounts of phospholipids is investigated as a possible mRNA delivery system for treating Inflammatory Bowel Diseases. Compared to LNPs with benchmark composition (b-LNPs), n-LNPs containing 30% distearoylphosphatidylcholine (DSPC) are well tolerated following intravenous administration and display natural targeting toward the inflamed colon in dextran sodium sulfate (DSS)-colitis bearing mice, while de-targeting clearing organs such as the liver and spleen. Using interleukin-10-encoding mRNA as therapeutic cargo, n-LNPs demonstrated a reduction of pathological burden in colitis-bearing mice. n-LNPs represent a starting point to further investigate the influence of LNPs composition on systemic biodistribution, ultimately opening new therapeutic modalities in different pathologies.

Polyinosinic/Polycytidylic Lipid Nanoparticles Enhance Immune Cell Infiltration and Improve Survival in the Glioblastoma Mouse Model.

Glioblastoma (GBM) immunotherapy is particularly challenging due to the pro-tumorigenic microenvironment, marked by low levels and inactive immune cells. Toll-like receptor (TLR) agonists have emerged as potent immune adjuvants but failed to show improved outcomes in clinical trials when administered as a monotherapy. We hypothesize that a combined nanoparticulate formulation of TLR agonist and immunogenic cell death-inducing drug (doxorubicin) will synergize to induce improved GBM immunotherapy. Lipid nanoparticle (LNP) formulations of the TLR agonists CpG and polyinosinic/polycytidylic (pIpC), with and without Dox, were first prepared, achieving an encapsulation efficiency >75% and a size <140 nm. In vitro studies identified that LNP pIpC was superior to CpG at activating bone marrow-derived immune cell populations (dendritic cells and macrophages) with minimal toxicity. It was also observed that the pIpC formulation can skew macrophage polarization toward the antitumorigenic M1 phenotype and increase macrophage phagocytosis of cancer cells. Upon intratumoral administration, pIpC Dox LNPs led to significant immune cell infiltration and activation. In survival models, the inclusion of Dox into pIpC LNP improved mice survival compared to control. However, addition of Dox did not show significant improvement in mice's survival compared to singly formulated pIpC LNP. This study has illustrated the potential of pIpC LNP formulations in prospective GBM immunotherapeutic regimes. Future studies will focus on optimizing dosage regimen and/or combination with other modalities, including the standard of care (temozolomide), immune checkpoint blockade, or cancer vaccines.

The influence of citrate buffer molarity on mRNA-LNPs: Exploring factors beyond general critical quality attributes

Lipid nanoparticles (LNPs) are crucial in delivering mRNA vaccines and therapeutics. The properties of LNPs can be influenced by the choice of lipids and the manufacturing conditions, such as mixing parameters, lipid concentration, and the type and concentration of the aqueous buffer used. In this study, we investigated the impact of the citrate buffer molarity, the buffer commonly used to dissolve mRNA in the preparation of mRNA-LNPs. We prepared SM-102 LNPs containing firefly luciferase mRNA using citrate buffers at molarities of 50 mM, 100 mM, or 300 mM. Our findings revealed that varying the molarity of the citrate buffer did not significantly affect the particle size when considering the average diameter (z-average or Mode). All formulations exhibited low polydispersity index (PDI) and high encapsulation efficiency. Detailed analysis of particle size sub-populations (D10, D50, and D90) and morphology indicated that citrate buffer concentration might influence lipid packing during LNP production, though these differences were subtle. However, using higher citrate molarity (300 mM) to produce LNPs notably reduced cellular internalisation and in vitro transfection efficiency. This trend was also observed in vivo, where similar expression levels were noted in mice receiving the 50 mM and 100 mM LNP formulations, but lower expression was seen for the 300 mM formulation. Our study highlights the importance of buffer molarity in the aqueous phase during mRNA-based LNP preparation and that generally reported critical quality attributes (CQAs) for LNPs may not detect subtle formulation differences.

DDEL-01. ENGINEERING MRNA THERAPIES FOR BRAIN TUMORS

Abstract Safe delivery of mRNA to the brain will revolutionize the treatment of brain tumors. While lipid nanoparticles (LNPs) are clinically most advanced non-viral delivery vehicles for therapeutic mRNA, LNP-mediated mRNA delivery to the brain remains challenging. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain cells without undue toxicity. We engineered LNPs consisting of four components similar to the formulation used in the mRNA COVID-19 vaccines (Moderna and Pfizer-BioNTech): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. We screened ten classes of helper lipids based on lipids enriched in extracellular vesicles to engineer biomimetic LNPs and tested their GFP mRNA delivery efficacy in SIM-A9 mouse microglia cell line. Several unique LNP formulations with potent delivery efficacy (&amp;gt;90% cells transfected) and stable GFP expression kinetics (5 days) were identified. LNP formulations with high transfection efficacy were then tested in vivo for luciferase mRNA delivery via intrathecal injection in C57BL/6 mice. Luciferase expression in vivo confirmed widespread mRNA delivery in the brain. We then tested Cre recombinase mRNA delivery in Ai9 mouse to identify LNP-targeted cells via flow cytometry and histology. Flow cytometry and expansion microscopy confirmed Cre recombinase mRNA delivery to a variety of brain cells, including microglia (75-90%), neurons (31-40%), neural stem cells (39-62%), oligodendrocytes (70-90%), and astrocytes (44-76%). LNPs were further evaluated for Cas9 mRNA and CD81 sgRNA delivery in C57BL/6 mouse brains to assess brain-targeted gene editing. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~40% of cells in the mouse brain. In summary, we engineered extracellular vesicle-based LNP library that can deliver RNA therapeutics to a variety of brain cells in vivo. With further development, this technology could potentially enable genetic and epigenetic therapies targeting drivers of brain tumors.

Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

Glycogen storage disease type-Ia patients, deficient in the G6PC1 gene encoding glucose-6-phosphatase-α, lack blood glucose control, resulting in life-threatening hypoglycemia. Here we show our humanized mouse model, huR83C, carrying the pathogenic G6PC1-R83C variant displays the phenotype of glycogen storage disease type-Ia and dies prematurely. We evaluate the efficacy of BEAM-301, a formulation of lipid nanoparticles containing a newly-engineered adenine base editor, to correct the G6PC1-R83C variant in huR83C mice and monitor phenotypic correction through one year. BEAM-301 can correct up to ~60% of the G6PC1-R83C variant in liver cells, restores blood glucose control, improves metabolic abnormalities of the disease, and confers long-term survival to the mice. Interestingly, just ~10% base correction is therapeutic. The durable pharmacological efficacy of base editing in huR83C mice supports the development of BEAM-301 as a potential therapeutic for homozygous and compound heterozygous glycogen storage disease type-Ia patients carrying the G6PC1-R83C variant.

(E, E)-farnesol and myristic acid-loaded lipid nanoparticles overcome colistin resistance in Acinetobacter baumannii

The rise of colistin-resistant Acinetobacter baumannii has severely limited treatment options for infections caused by this pathogen. While terpene alcohols and fatty acids have shown potential to enhance colistin’s efficacy, but their high lipophilicity limits their clinical application. To address this, we developed water-dispersible lipid nanoparticles (LNPs) in two sizes (40 nm and 130 nm), loaded with these compounds to act as colistin adjuvants. Among eleven LNP formulations, six significantly reduced colistin’s minimum inhibitory concentration (MIC) by 16- to 64-fold. The most effective, featuring (E,E)-farnesol and myristic acid, were further examined for bactericidal activity, membrane disruption, cytotoxicity, and in vivo efficacy in Galleria mellonella larvae. Time-kill studies demonstrated that at an adjuvant concentration of 60 mg/L, these LNPs eradicated bacteria when combined with 4 mg/L free colistin for resistant isolates (MIC = 128 mg/L) and 0.06 mg/L for susceptible isolates (MIC = 0.5 mg/L), without regrowth. Myristic acid-loaded LNPs combined with free colistin at 1/8 MIC resulted in a 4.2-fold higher mortality rate than the combination with (E,E)-farnesol-loaded LNPs in resistant strains. This result was correlated with a 45-fold faster increase in inner membrane permeability, measured by propidium iodide (PI) uptake, in the presence of myristic acid-loaded LNPs compared with a 13-fold faster increase with (E,E)-farnesol-loaded LNPs. DiSC3(5) assays revealed that LNPs alone depolarised the bacterial inner membrane, with enhanced effects when combined with colistin at 1/8 MIC, a result not observed with colistin alone at this concentration. As with PI uptake, this inner membrane depolarising effect was more pronounced with myristic acid-loaded LNPs than with (E,E)-farnesol-loaded LNPs in resistant strains, suggesting that the colistin adjuvant effect of these lipophilic compounds is due to their ability to help colistin destabilise the bacterial inner membrane. Cytotoxicity assays demonstrated no adverse effects on bone marrow macrophages after 6 h of exposure, although some toxicity was observed after 24 h. No mortality was observed in Galleria mellonella larvae over 7 days following three consecutive days of treatment with colistin and LNPs. Notably, the combination of (E,E)-farnesol-loaded LNPs and colistin significantly improved the survival of Galleria infected with A.baumannii. These results suggest that lipophilic-adjuvant-loaded LNPs may offer a promising strategy to enhance colistin efficacy and combat antibiotic-resistant A. baumannii infections.

Enhanced Broad-Spectrum Efficacy of an L2-Based mRNA Vaccine Targeting HPV Types 6, 11, 16, 18, with Cross-Protection Against Multiple Additional High-Risk Types.

Current L1-based human papillomavirus (HPV) vaccines provide type-specific protection but offer limited cross-protection against non-vaccine HPV types. Therefore, developing a broad-spectrum HPV vaccine is highly desirable. In this study, we optimized mRNA constructs and developed a multivalent L2-based mRNA vaccine encoding L2 aa 2-130, which includes all known neutralizing epitopes from four prevalent HPV types (HPV-6, -11, -16, and -18). We evaluated its immunogenicity in a mouse model and compared the efficacy of a commercially available mRNA delivery reagent with a custom-synthesized lipid nanoparticle (LNP) formulation. We identified that a construct containing E01 (a 5'-untranslated region) and SL2.7 (a poly(A) polymerase recruitment sequence) significantly increased protein expression. The L2-based mRNA vaccine induced robust and long-lasting humoral immune responses, with significant titers of cross-reactive serum IgG antibodies against L2 epitopes. Notably, the vaccine elicited cross-neutralizing antibodies and conferred cross-protective immunity not only against vaccine-targeted HPV types but also against non-vaccine HPV types, following intravaginal challenge in mice. We also found that LNP delivered mRNA more effectively in vivo. The L2-based mRNA vaccine developed in this study shows significant potential for broad-spectrum protection against multiple HPV types. This approach offers a promising strategy for reducing the global burden of HPV-associated cancers.

Optimizing lipid nanoparticles for fetal gene delivery in vitro, ex vivo, and aided with machine learning

There is a clinical need to develop lipid nanoparticles (LNPs) to deliver congenital therapies to the fetus during pregnancy. The aim of these therapies is to restore normal fetal development and prevent irreversible conditions after birth. As a first step, LNPs need to be optimized for transplacental transport, safety on the placental barrier and fetal organs and transfection efficiency. We developed and characterized a library of LNPs of varying compositions and used machine learning (ML) models to delineate the determinants of LNP size and zeta potential. Utilizing different in vitro placental models with the help of a Random Forest algorithm, we could identify the top features driving percentage LNP transport and kinetics at 24 h, out of a total of 18 input features represented by 41 LNP formulations and 48 different transport experiments. We further evaluated the LNPs for safety, placental cell uptake, transfection efficiency in placental trophoblasts and fetal lung fibroblasts. To ensure the integrity of the LNPs following transplacental transport, we screened LNPs for transport and transfection using a high-throughput integrated transport-transfection in vitro model. Finally, we assessed toxicity of the LNPs in a tracheal occlusion fetal lung explant model. LNPs showed little to no toxicity to fetal and placental cells. Immunoglobin G (IgG) orientation on the surface of LNPs, PEGylated lipids, and ionizable lipids had significant effects on placental transport. The Random Forest algorithm identified the top features driving LNPs placental transport percentage and kinetics. Zeta potential emerged in the top driving features. Building on the ML model results, we developed new LNP formulations to further optimize the transport leading to 622 % increase in transport at 24 h versus control LNP formulation. To induce preferential siRNA transfection of fetal lung, we further optimized cationic lipid percentage and the lipid-to-siRNA ratio. Studying LNPs in an integrated placental and fetal lung fibroblasts model showed a strong correlation between zeta potential and fetal lung transfection. Finally, we assessed the toxicity of LNPs in a tracheal occlusion lung explant model. The optimized formulations appeared to be safe on ex vivo fetal lungs as indicated by insignificant changes in apoptosis (Caspase-3) and proliferation (Ki67) markers. In conclusion, we have optimized an LNP formulation that is safe, with high transplacental transport and preferential transfection in fetal lung cells. Our research findings represent an important step toward establishing the safety and effectiveness of LNPs for gene delivery to the fetal organs.

Enhancing RNA encapsulation quantification in lipid nanoparticles: Sustainable alternatives to Triton X-100 in the RiboGreen assay

To quantify concentration and encapsulation efficiency (EE) of mRNA in lipid nanoparticles (LNPs) the RiboGreen assay is extensively used. As part of this assay, a surfactant is used to release mRNA from LNPs for detection with the RiboGreen dye. So far, the surfactant of choice has been Triton X-100, which is harmful to human health and the environment. Alternatives to Triton X-100 are therefore needed, but surprisingly no such effort has yet been described in the literature. Here we show how three, less harmful, surfactants (Brij 93, Zwittergent 3–14 and Tween 20) compare to Triton X-100 for releasing mRNA from LNPs for detection with the RiboGreen assay. We found that Zwittergent 3–14 and Tween 20 at high concentrations (0.5 %) are at the minimum as effective as Triton X-100 at high concentration (0.5 %) across three different mRNA-LNP formulations. Interestingly, Tween 20 was the most effective at releasing mRNA from LNPs, across all concentration ranges explored (0.0025 %, 0.01 %, 0.1 % and to 0.5 % (v/v)) highlighting its potency at solubilizing the three different LNP formulations. Our results show that Tween 20 can be used as an alternative to Triton X-100 in the RiboGreen assay, resulting in more accurate quantification of the total mRNA concentration and EE%, as well as making the assay more environmentally friendly. Such improvement could potentially increase the likelihood of identifying therapeutically attractive hard-to-solubilize LNP-mRNA formulations that would be discharged when using Triton X-100 due to their apparent low EE values, as well as ensure more accurate mRNA dosing in both in vitro and in vivo studies.

Encapsulation of Dexamethasone into mRNA–Lipid Nanoparticles Is a Promising Approach for the Development of Liver-Targeted Anti-Inflammatory Therapies

The objective of this study was to develop two lipid nanoparticle (LNP) formulations capable of efficiently expressing a reporter mRNA while co-delivering the anti-inflammatory drug dexamethasone (DX) to reduce inflammatory side effects in protein replacement therapies. Two types of LNPs were developed, in which 25% of cholesterol was replaced by DX. These LNPs contained either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a helper lipid. The resulting LNPs exhibited high stability, homogeneity, and near-neutral Zeta potentials. SAXS experiments confirmed DX incorporation into the LNP core, with slow in vitro DX release observed over 48 h. The LNPs achieved high mRNA encapsulation efficiency (95–100%) and effectively transfected HepG2 cells, dendritic cells, and hPBMCs. While LNPs increased cytokine release (IL-1β, TNF-α, MCP-1), LNPs-DX significantly reduced cytokine levels, demonstrating enhanced anti-inflammatory properties while maintaining mRNA expression levels. In vivo biodistribution showed predominant liver localization post-intramuscular injection, regardless of the DSPC or DOPE composition. LNPs co-loaded with mRNA and DX are promising candidates for continuous protein replacement. Due to their ability to reduce treatment-related inflammation while maintaining significant mRNA expression levels, these LNPs are perfectly suited for the treatment of liver-related metabolic diseases.