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DNA tetrahedra (DNA Td) are promising nanocarriers for drug delivery, but how hypoxia affects their cellular internalisation remains poorly understood. We synthesised and characterised Cy5-labelled DNA Td and established chemical hypoxia; in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1; nuclear translocation. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia, whereas transferrin and cholera toxin B uptake increased, indicating cargo-selective regulation. Temperature-arrest experiments confirmed reduced energy-dependent internalisation. Pharmacological profiling showed a shift from clathrin-mediated and galectin/glycan-dependent pathways toward lipid raft/cholesterol-dependent uptake. Hypoxia increased plasma membrane electronegativity, suggesting a biophysical barrier to DNA Td uptake. Importantly, DOTMA complexation restored uptake to normoxic levels, identifying electrostatic repulsion as a key determinant. In zebrafish larvae, hypoxia significantly enhanced whole-larva DNA Td accumulation. These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.

Membrane tension, defined as the energy cost to expand the membrane surface area, is a fundamental mechanical property of the lipid bilayer and an important modulator of various cellular processes. Liposomes provide model systems to study protein-mediated membrane dynamics, where membrane tension can be controlled by osmotic pressure and micropipette aspiration. However, it is challenging to individually control membrane tension of nanometer-sized liposomes, such as small unilamellar vesicles (SUVs), that are widely used for modeling subcellular membrane structures and drug delivery. To bridge this technological gap, we present reconfigurable DNA origami rings for dynamically controlling membrane tension of sub-100 nm liposomes. The DNA nanorings template the formation of uniformly sized SUVs and undergo trigger-responsive dilation and contraction, thus applying controlled and reversible mechanical stress to the SUV membranes. Electron microscopy analyses show deformed liposomes with ~2.5% expanded membrane area following ring dilation. The corresponding increase in membrane tension opens a mechanosensitive ion channel MscL with gating tension of ~10 mN/m. Selectively manipulating a mixture of SUV populations enables timed release of their distinct molecular cargos, either concurrently or one at a time. Furthermore, vesicles with expanded membranes are more conducive to SNARE-mediated fusion. The nanomechanical device thus provides a programmable platform for engineering the mechanics of synthetic nano-vesicles and studying membrane tension modulated processes at the molecular level.

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

Endosomal escape remains the central bottleneck limiting lipid nanoparticle (LNP)-mediated mRNA delivery. In this study, we use giant unilamellar vesicles (GUVs) mimicking the endosomal membrane to directly visualize the interaction and membrane disruptions caused by two model LNPs constituted by two different ionizable lipids: LP01 and BiP-20 (a Branched ionizable Phospholipid) that are both benchmark ionizable lipids for in vivo gene editing in liver. Despite identical TNS-measured apparent pKa (6.6) for both lipids in our conditions, LP01 LNP showed maximal membrane engagement at pH 5.8, while BiP20 LNP required pH 5.1, demonstrating that bulk pKa alone cannot predict the pH at which functional membrane interaction occurs. Using confocal microscopy, we show that the LNP engagement with the endosomal membrane proceeds through first electrostatic attraction, clustering, membrane disruption by forming highly curved morphologies, and finally leading to the rupture of the whole endosomal mimicking membrane. Notably, the mRNA cargo is released only upon complete lysis of the membrane. This indicates that the membrane destabilization and cargo release are mechanistically distinct steps. These findings establish a cell-free platform for dissecting individual sub-steps of endosomal escape.

Conducting polymers are widely used in bioelectronic interfaces because of their mixed ionic-electronic conductivity, mechanical compliance, and compatibility with biological systems. However, their electrochemically driven structural dynamics have received little attention as a mechanism for mechanical cell stimulation. Here, we show that electrochemical actuation of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) generates mechanical cues capable of activating endogenous mechanosensitive pathways in HEK293T cells. Transparent PEDOT:PSS films deposited on ITO exhibited a heterogeneous granular morphology and underwent potential-dependent microscopic deformation during electrochemical modulation. Direct optical tracking revealed displacement of the polymer boundary, with structural changes occurring preferentially in polymer-dense regions and propagating toward the film edge. When HEK293T cells were cultured directly on PEDOT:PSS, repeated electrochemical stimulation at -240 mV produced reproducible intracellular Ca2+ responses. Pharmacological inhibition with GsMTx4 attenuated the calcium response, whereas blockade of voltage-gated sodium channels with tetrodotoxin largely preserved it, supporting the involvement of mechanosensitive pathways in the cellular response. These findings identify PEDOT:PSS as an electromechanical biointerface in which electrochemical modulation can introduce a mechanical component alongside the established electrical function of the interface. This mechanical contribution should therefore be considered when interpreting cellular responses to conducting polymer- based electrical stimulation and provides a basis for engineering bioelectronic interfaces that deliberately couple electrical control with mechanotransduction.

Capillaric circuits (CCs) enable pre-programmed liquid handling through self-filling and passive valving governed by capillary forces, eliminating the need for external pumps and actuators. However, CCs optimized for high cohesion liquids (HCL) such as water are intrinsically unsuitable for programmed flow control of low cohesion liquids (LCL) such as oils and solvents because the high surface free energy needed for capillary flow of HCL results in uncontrolled, complete wetting by LCL. Here, we introduce a library of multiphase components including phase-to-phase valves (P2PV), hydro-pneumatic relays (HPR) and multiphase domino valves (MDVs) that collectively enable CCs to concomitantly process HCLs and LCLs. P2PVs use a pre-filled HCL to valve immiscible LCLs (e.g. oil) by confining the LCL, and upon triggering, hydraulically entrain it. To prevent uncontrolled mixing between miscible LCLs (e.g. ethanol) and the HCL, HPRs with an air gap and air waste are added to the P2PV. MDVs are further added and enable preprogrammed, sequential delivery of LCL and HCL by multiphase microfluidic chain reactions. Multiphase liquid processing is applied to automated, on-chip lipid nanoparticle (LNP) manufacturing, illustrating the potential of multiphase CCs.

Despite their promise, lipid nanoparticle gene delivery systems have repeatedly failed clinical trials and struggle to achieve efficient, localized transfection in target tissues. The majority of endocytosed nanoparticles are degraded before nucleic acid release, and an inability to track particle distribution in vivo prevents validation of successful delivery. Alternatively, nanobubbles (NBs) are lipid-shelled, gas-core preclinical ultrasound contrast agents and stimuli-responsive drug delivery vehicles. Under varying acoustic pressures, NBs expand, contract, and burst, releasing cargo in an externally controlled, site-specific manner while scattering unique echoes for simultaneous ultrasound visualization. Here, we introduce a cationic nanobubble (CNB) formulation with a +42.3 mV zeta potential, 265 nm diameter, and 2.43x1011 NBs/mL concentration. CNBs produce stable ultrasound contrast, electrostatically load plasmid DNA onto their surface, and internalize into >99% of human prostate cancer cells within 15 minutes in vitro. CNBs remain brightly echogenic intracellularly and induce sonication-dependent expression of green fluorescent protein (GFP). In vivo, CNBs generate contrast in mouse livers for 50 minutes after intravenous administration. Therapeutic ultrasound stimulation over the liver causes a sharp reduction in ultrasound contrast, visualizing localized cavitation in the target organ and inducing a 2.5-fold increase in anti-GFP mean fluorescence intensity relative to the untransfected control. Importantly, no GFP expression is observed without ultrasound stimulation, supporting a mechanism for selective and site-specific gene delivery. This study presents a highly stable CNB capable of efficient DNA loading and ultrasound-dependent gene expression. These results provide a foundation for the future development of CNB platforms to advance image-guided, ultrasound-triggered gene therapy.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

Subcellular thermal engineering provides a powerful approach for investigating and manipulating biological processes. However, existing subcellular heating platforms capable of combining spatially confined heating, quantitative thermometry and simultaneous imaging of cellular responses remain limited. We developed a quantitative nanoheater-thermometer (qNanoHT), a polymeric nanoparticle integrating a temperature-sensitive fluorescent, dye and a photothermal dye. qNanoHT determines local temperature from fluorescence lifetime using fluorescence lifetime imaging microscopy (FLIM), thereby reducing susceptibility to photobleaching, focal drift and variations in probe concentration compared with intensity-based methods. The platform enabled real-time measurement at a subcellular heat spot while the dynamics of heat shock factor 1 (HSF1) were monitored in living cells. Heating at a single intracellular site was sufficient to induce HSF1 foci. Foci induced by mild heating at approximately 38 {degrees}C dissolved after heating ceased, whereas those induced by stronger heating at approximately 41 {degrees}C persisted and were associated with caspase-3/7 activation and apoptosis. Notably, qNanoHT-mediated subcellular heating induced HSF1 foci at a lower measured temperature than uniform whole-cell heating approximately 38 {degrees}C versus 39 {degrees}C indicating that the spatial extent of heating influences the HSF1 activation threshold. qNanoHT therefore provides a quantitative platform for relating local intracellular temperature to cellular stress responses and subsequent cell fate.

Nanobubbles (NBs) have been widely investigated as ultrasound contrast and therapeutic agents, yet whether submicron gas bubbles can directly generate measurable ultrasound signals remains controversial. Because acoustic scattering scales with bubble size, several studies have recently attributed nanobubble echogenicity primarily to larger bubbles. A recent Science Advances publication, attributed the loss of ultrasound signal after filtration to the removal of residual larger bubbles [1]. This interpretation assumes that filtration acts solely as a size-selection process. Here, we show that filtration also alters the shell properties and stability of lipid-coated NBs, independent of size. Filtered NBs (F-NBs) show significant lipid loss and accumulation on the filters themselves, resulting in decreased bubble stability and greater susceptibility to destruction under acoustic excitation. To decouple size from processing effects, we generated NB populations size-matched to filtered samples using increased centrifugation (IC-NBs), a comparatively gentler separation method. Despite comparable size distributions, IC-NBs retained sustained ultrasound scattering, whereas filtered NBs showed significantly reduced acoustic persistence. Filtration through 5 m pores, ~5-fold larger than the largest bubbles, preserved acoustic persistence but still reduced bubble concentration by ~30%, suggesting that membrane contact may contribute to bubble loss beyond size exclusion. Complementary lipid and gas-content assays provided further evidence of filtration-induced changes. The 0.45 m filtration removed substantially more total lipid than could be attributed to free-lipid removal alone, while the lipid associated with the larger bubbles removed during filtration accounted for only ~5% of the additional lipid loss. Encapsulated gas content remained comparable to that of size-matched centrifuged controls, suggesting that the reduced acoustic stability of filtered NBs is more closely associated with changes in lipid content than with loss of the gas core. These results indicate that membrane filtration is not a purely size-selective process: it removes substantially more lipid than size exclusion predicts and yields bubbles that are less stable under ultrasound, most likely through changes in shell lipid content or in the surrounding lipid reservoir, underscoring the importance of processing-induced effects in interpreting nanobubble echogenicity.

Four-dimensional (4D) materials incorporating functional gradient designs offer a powerful platform for engineering dynamic structures capable of programmed shape transformations in response to environmental stimuli. However, most gradient-based 4D systems rely on uniaxial gradients, which typically generate simple, symmetric deformations with uniform curvature, limiting their ability to recreate biomimetic architectures that require spatially coordinated morphogenesis. Here, we report a biaxial gradient-engineered 4D hydrogel system capable of programmable, non-uniform shape morphing within a single construct. A one-step photocrosslinking strategy integrates vertical light attenuation and horizontal grayscale photomask patterning to establish orthogonal crosslinking gradients along two directions, producing spatially heterogeneous swelling stresses that drive controlled multi-directional deformation. The resulting hydrogels exhibit tunable swelling and mechanical properties, enabling precise regulation of curvature distribution and shape transformation. This biaxial gradient platform generates diverse biomimetic architectures, including swan-neck, fiddlehead fern, sea star, and Euonymus europaeus-like structures. Importantly, the system supports cell-laden biofabrication, where human mesenchymal stem cell-encapsulated constructs maintain high viability and undergo chondrogenic differentiation while preserving programmed morphologies. This work establishes biaxial gradient-programmed 4D hydrogels as a robust strategy for integrating morphogenesis with tissue formation, advancing biomimetic biofabrication and morphogenetic tissue engineering.

Gold electrodes are attractive substrates for bioelectronic and cell-free synthetic biology platforms because they are conductive, chemically stable, biocompatible, and readily functionalized through thiol-gold chemistry. Here, gene-length DNA monolayers assembled on planar gold electrodes as reusable templates for cell-free protein expression are investigated. Using thiol-modified sfGFP genes, DNA surface density is shown to be tunable by changing the DNA concentration during incubation, with the immobilized genes able to support cell-free sfGFP expression directly from the electrode surface. Further, the effects of applied voltage, storage, repeated reactions, reducing agents, and protein fouling on monolayer stability and expression output are examined. While some conditions lead to loss of reusable expression activity, dense chemisorbed monolayers can retain partial function under neutral, non-reducing conditions and are relatively robust to protein exposure. In contrast, low-density physisorbed monolayers show a stronger relationship between DNA loss and expression output. Finally, when using gold-mediated fluorescence quenching to monitor changes in DNA conformation, surface-bound DNA demonstrates electrophoretic addressability. Together, these results establish DNA- functionalized planar electrodes as a promising foundation for modular, addressable cell-free expression platforms.

Cell fusion enables cell-based applications, yet systematic optimization remains constrained by low throughput (microfluidic devices) and manual operation (bulk protocols). Here, an automated microfluidic platform is presented for cell pairing and chemical fusion at a statistically relevant scale. High throughput is achieved by arranging ~350,000 deformability-based cell traps as concentric circular arrays in a radial chip. Using tumor and dendritic cells as a model for cancer vaccine, the device reproducibly co-occupies ~75% of traps (~250,000) of which ~140,000 hold exactly one cell of each type. This exceeds the yield of previously reported in hydrodynamic pairing arrays by more than an order of magnitude. A concentration step-up-and-down PEG profile yielded a fusion efficiency of ~20% (range 17.7-23.4%, n =3) at viability above 90% that results in producing at least ~50,000 hybrids per chip. This efficiency is comparable to that reported for PEG-based fusion in small-scale microfluidic devices but obtained at higher throughput and approximately four-fold higher than the conventional benchtop protocol. Fusion efficiencies were quantified by image analysis and validated by flow cytometry. By combining automation, large-area circular trap arrays and reagent control, the platform is useful for data-rich optimization of cell engineering processes previously constrained by limited microfluidic throughput.

Achieving precise spatiotemporal control over neuropeptide delivery in vivo remains a major challenge, as conventional approaches lack temporal resolution and control over release kinetics or have limitations for in vivo applications. Here, we demonstrate that photoswitchable azobenzene-containing lipid nanovesicles ("azosomes") enable light-controlled release of neuropeptides in vivo in the brain of awake moving mice. Azosomes were infused into the hippocampus via optofluidic cannulas and activated using light stimulation in freely behaving mice. In vivo release kinetics were systematically characterized using calcein-loaded azosomes by varying light power, pulse duration, and post-infusion time. Oxytocin (OT)-loaded azosomes were used to assess bioactivity and receptor specificity using dual-color fiber photometry with the genetically encoded OT sensor (MTRIAOT), alongside pharmacological blockade with the oxytocin receptor antagonist OVTA (Ornithine-VasoTocin Analog). Azosomes enabled robust, repeatable, and light-dependent cargo release in vivo with tunable kinetics governed by stimulation parameters, with release efficiency controlled by light power and pulse duration. The system maintained functional stability for several hours post-infusion, with near-complete release achievable within a [~]2-hour window and minimal baseline leakage prior to stimulation. Controlled OT delivery produced rapid, receptor-specific increases in MTRIAOT signals and modulated CA2 hippocampal circuit activity, reducing excitatory neuronal transient frequency and amplitude while altering social interaction dynamics, including decreased latency to initiate contact. These findings establish, for the first time, photoswitchable nanovesicles as a versatile platform for spatiotemporally precise delivery of neuropeptides in vivo, overcoming key limitations of existing delivery strategies and providing a broadly applicable framework for manipulating neuromodulatory signaling with high temporal precision.

Living cells harbor numerous membraneless organelles (MOs), which are dynamic protein/nucleic acid assemblies responding to specific physicochemical triggers. Liquid-liquid phase separation (LLPS) plays a crucial role in their formation and activity. Inspired by the natural MOs that maintain their individual identities, this work presents a bioengineering strategy to generate LLPS-driven, isochemical MO populations using surfactant-like peptides that stabilize the MO interface. The result is highly stable, monodispersed, sub-{micro}m-sized MO populations, which are not only capable of compartmentalizing synthetic cells but also provide superior environments for enzymatic reactions. This is achieved using pH-responsive elastin-like polypeptides (PREs) as MOs and formulating an amphiphilic PRE-based peptide to stabilize the MO interface. Relative abundance of the surface-active peptide, as well as the rate of pH change, allows direct control over the MO size. Encapsulating these components within synthetic vesicles using a microfluidic platform leads to on-demand multi-compartmentalization via an external pH trigger. Lastly, a functional consequence of the acute size control is shown through a phosphatase reaction, where the highest reaction rate is observed in size-controlled MOs when compared to dilute environments and surfactant-free MOs. The presented strategy provides a new avenue for designing programmable MOs and thus achieve functional compartmentalization within synthetic cells.

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

The nuclear pore complex (NPC) is a massive protein system that controls all nucleocytoplasmic transport via a dynamic network of intrinsically disordered proteins called FG-Nups. We developed an NPC-mimicking DNA origami nanostructure that self-assembles into a tetrameric, octagonal nanopore with an inner diameter that, through addition of DNA oligonucleotides, can be tuned in a user-defined fashion from 57 to 66 nm - mimicking the contracted and dilated states of NPCs. We demonstrated that these origami nanopores can dilate within minutes and recontract at slower timescales. Both contracted and dilated pores can be inserted into lipid bilayers, where they maintain their conformation and allow transmembrane transport of fluorescent proteins. With outer diameters up to 87 nm, these are, to our knowledge, the largest DNA origami nanopores inserted into lipid membranes. Functionalization of the pores with the FG-Nup Nsp1 reduces non-specific cargo diffusion across the lipid membrane while facilitating translocation of the transport receptor Kap95, demonstrating transport selectivity much like biological NPCs. The work establishes a size-tunable platform to dissect nuclear transport mechanisms, with potential applications for engineering programmable artificial channels.

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

Understanding the physicochemical factors that govern siRNA nanocarrier assembly is essential for the rational design of effective delivery systems. By optimizing various lipid compositions, cholesterol content and PEG length we created a peptide-functionalized cationic liposomal platform made of DOPE/TAP lipids with cholesterol-anchored nona-arginine (R9-Chol) for siRNA complexation, intracellular transport and effective silencing of the target EGFR gene. Analysis of {zeta}-potential and dynamic light scattering allowed to rationally design formulation of stable, monodisperse nanoscale lipoplexes with a positive surface charge. With fluorescence polarization, circular dichroism and agarose gel electrophoresis we found an optimal siRNA:liposome complexation ratio of 1:77, which protected siRNA from ribonuclease-mediated degradation. Morphological imaging confirmed a shift from discrete vesicular structures to organized multilamellar lipoplexes, consistent with electrostatically driven self-assembly. In cellular studies, the optimized nanocarrier promoted efficient uptake of fluorescent siRNA in MDA-MB-231 cells and achieved functional delivery of anti-EGFR, leading to substantially reduced expression of the target gene at both transcript and protein levels. This work offers mechanistic understanding of peptide-assisted lipid:siRNA assembly and positions R9-functionalized DOPE/TAP liposomes as a promising platform for siRNA delivery.