Client Citation Analysis
The Dropometer provided interfacial tension measurements used to compare lipid formulations and inform emulsification conditions during asymmetric vesicle assembly. It was not the primary measurement instrument; principal outcomes (transfection efficiency, cellular uptake, gene editing) were measured by confocal microscopy, flow cytometry, and sequencing.
Interfacial tension between mineral oil and PBS containing DODMA, EPC, POPC, and POPS lipid formulations, measured by pendant drop with Young–Laplace fitting. Data appear in Figure 3b–c.
"Pendant drop method (Droplet Lab)" — Experimental Section. Lipid dissolved in mineral oil at 0.06 mM; oil droplet contour analysed with Young–Laplace fit.
To compare five conditions at the water/oil interface. A correlation was observed between lower interfacial tension and higher asymmetric vesicle yield, informing the formulation selected for drug delivery experiments.
Mean ± SD, n = 3 : consistent with standard pendant-drop methodology for lipid/oil systems.
Interfacial tension between mineral oil and 1× PBS was measured for five conditions: DODMA, EPC, POPC, and POPS (each at 0.06 mM in mineral oil), plus a no-lipid baseline. An oil droplet was introduced into aqueous PBS; the droplet contour was imaged and fitted with the Young–Laplace equation to determine interfacial tension. Only DODMA carries an explicitly stated SD in the paper text (61.5 ± 3.5 mN/m); values for EPC, POPC, and POPS are read from Figure 3c error bars. Mean ± SD, n = 3.
Hydrodynamic vesicle diameter by DLS (Figures 1b, 2c)
Zeta potential, surface charge (Figures 2a–b, 2d)
Confocal microscopy: uptake, transfection, protein delivery (Figures 4–6)
Flow cytometry: cellular uptake quantification (Figure 4b–c)
Fluorescence intensity: vesicle yield and concentration
Laurdan fluorescence: membrane fluidity and vesicle stiffness (Figure S13)
qRT-PCR: EGFR silencing by siRNA delivery (Figure S16)
Next-generation sequencing: Cas9/sgRNA gene-editing efficiency
Transmission electron microscopy: vesicle size validation alongside DLS
The Dropometer was used to measure interfacial tension between mineral oil and PBS solutions containing different lipid compositions. In the pendant drop workflow, lipid-containing mineral oil droplets were introduced into aqueous PBS using a syringe needle. The droplet contour was imaged and analysed with a Young–Laplace fit to determine interfacial tension. Five conditions were compared: DODMA, EPC, POPC, and POPS (each at 0.06 mM in mineral oil) and a no-lipid baseline.
The resulting interfacial tension values were used to identify a correlation between lipid-dependent interfacial behaviour and asymmetric vesicle yield during the inverted emulsion assembly process. POPS-containing systems, which showed the lowest interfacial tension, also produced the highest vesicle yields. The authors describe this as an observed correlation; the underlying mechanism was not mechanistically tested in this study.
POPS produced the lowest interfacial tension (approx. 12 mN/m) and the highest asymmetric vesicle yield. The paper describes this as an observed correlation; the underlying mechanism was not mechanistically tested.
POPC-POPS vesicles showed approximately twofold higher uptake than symmetric POPS-POPS vesicles. Inverting the composition to POPS-POPC reduced uptake fourfold relative to POPC-POPS, showing that which lipid occupies which leaflet matters independently of overall lipid identity.
POPC-POPS vesicles achieved transfection rates 9× higher than POPC-POPC controls and 7× higher than POPS-POPS. Optimised commercial LNPs remain approximately 50% efficient against these vesicles in this study an important benchmark gap the authors acknowledge.
POPC-POPS vesicles showed lower LDH-measured cytotoxicity than symmetric POPS-POPS vesicles. The authors hypothesise this reflects resemblance to the natural cell membrane, which contains PS in the inner leaflet, but the mechanism was not elucidated.
Multiple proteins (60–240 kDa) were delivered to HEK293 cells with confirmed cytoplasmic release. NLS-tagged Cas9/sgRNA produced TTR gene edits in 7% of HEK293 and 9% of HeLa cells. The lipofectamine benchmark stands at 82% a gap the authors explicitly flag for future optimisation.
Figures shown as reference thumbnails only. Full figures available at the publisher's page via DOI links below. © 2025 Wiley-VCH GmbH.
Pendant drop workflow and interfacial tension values for five conditions: the Dropometer data.
Inverted emulsion workflow; vesicle asymmetry (>90%) and size control validated by DLS and dithionite quenching.
Confocal and flow cytometry comparison across four vesicle compositions including the inverted POPS-POPC condition.
GFP expression post mRNA delivery; transfection efficiency and cytotoxicity comparisons across vesicle types.
All limitations below are drawn directly from the authors' own discussion and conclusions.
The relationship between lower interfacial tension and higher vesicle yield is empirical. The paper notes this as "an interesting correlation" but does not propose or test a molecular mechanism.
Cas9/sgRNA editing achieved 7–9% efficiency against lipofectamine's 82%. The authors explicitly flag this as a proof-of-concept result requiring further optimisation before clinical relevance.
All experiments were conducted in HEK293 and HeLa cell lines only. The authors identify targeting strategies and in vivo testing as necessary future steps.
The authors note reduced cytotoxicity for POPC-POPS and state it would be "particularly interesting to identify the exact molecular mechanisms" ; this remains an open question in the paper.
Asymmetric vesicle mRNA transfection efficiency is lower than optimised commercial LNPs (~50% for LNPs vs the values reported here). The authors state further optimisation is needed and that LNPs offer less compositional flexibility by comparison.
What molecular properties of POPS drive the lower interfacial tension, and can this be leveraged predictively to design higher-yield lipid systems without empirical screening?
Does asymmetric leaflet composition affect endosomal escape efficiency independently of cellular uptake, and through what biophysical mechanism?
Can gene-editing efficiency be increased to clinically relevant levels through lipid composition optimisation alone, or are additional endosomal escape strategies required?
Do these results translate to primary cell types and in vivo systems, where serum protein adsorption and immune clearance will alter vesicle behaviour?
Pendant-drop measurements can distinguish lipid systems before committing to full vesicle assembly runs, reducing wasted reagent and time at the formulation stage.
Zeta potential is controlled exclusively by the outer leaflet. Inner leaflet lipids can be varied independently to tune biophysical properties without changing surface charge.
The same lipids arranged asymmetrically in opposite orientations produce fourfold differences in cellular uptake; a parameter unavailable in conventional symmetric vesicle systems.
Shorter fatty acid chains produce softer, more fluid membranes with higher cellular uptake, suggesting membrane mechanics as an additional tuning lever beyond lipid identity.
The inverted emulsion method encapsulated mRNA, siRNA, and proteins from 60–240 kDa, including functional Cas9/sgRNA ribonucleoprotein complexes.