Cytotoxicity and cell membrane interactions of choline-based ionic liquids: Comparing amino acids, acetate, and geranate anions.

Ionic liquids (ILs) have found diverse applications in research and industry. Biocompatible ILs, a subset considered less toxic than traditional ILs, have expanded their applications into biomedical fields. However, there is limited understanding of the toxicity profiles, safe concentrations, and underlying factors driving their toxicity. In this study, we investigated the cytotoxicity of 13 choline-based ILs using four different cell lines: Human dermal fibroblasts (HDF), epidermoid carcinoma cells (A431), cervical cancer cells (HeLa), and gastric cancer cells (AGS). Additionally, we explored the haemolytic activity of these ILs. Our findings showed that the cytotoxic and haemolytic activities of ILs can be attributed to the hydrophobicity of the anions and the pH of the IL solutions. Furthermore, utilising quartz crystal microbalance with dissipation (QCM-D), we delved into the interaction of selected ILs, including choline acetate [Cho][Ac] and choline geranate [Cho][Ge], with model cell membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The QCM-D data showed that ILs with higher toxicities exhibited more pronounced interactions with membranes. Increased variations in frequency and dissipation reflected substantial changes in membrane fluidity and mass following the addition of the more toxic ILs. Furthermore, total internal reflection fluorescence microscopy study revealed that [Cho][Ac] could cause lipid rearrangements and pore formation in the membrane, while [Cho][Ge] disrupted the bilayer packing. This study advances our understanding of the cellular toxicities associated with choline-based ILs and provides valuable insights into their mechanisms of action concerning IL-membrane interactions. These findings have significant implications for the safe and informed utilisation of biocompatible ILs in the realm of drug delivery and biotechnology.

Cubosome lipid nanocarriers for delivery of ultra-short antimicrobial peptides.

HYPOTHESIS: Although antimicrobial peptides (AMPs) are a promising class of new antibiotics, their inherent susceptibility to degradation requires nanocarrier-mediated delivery. While cubosome nanocarriers have been extensively studied for delivery of AMPs, we do not currently understand why cubosome encapsulation improves antimicrobial efficacy for some compounds but not others. This study therefore aims to investigate the link between the mechanism of action and permeation efficiency of the peptides, their encapsulation efficacy, and the antimicrobial activity of these systems. EXPERIMENTS: Encapsulation and delivery of Indolicidin, and its ultra-short derivative, Priscilicidin, were investigated using SAXS, cryo-TEM and circular dichroism. Molecular dynamics simulations were used to understand the loading of these peptides within cubosomes. The antimicrobial efficacy was assessed against gram-negative (E. coli) and gram-positive (MRSA) bacteria. FINDINGS: A high ionic strength solution was required to facilitate high loading of the cationic AMPs, with bilayer encapsulation driven by tryptophan and Fmoc moieties. Cubosome encapsulation did not improve the antimicrobial efficacy of the AMPs consistent with their high permeation, as explained by a recent 'diffusion to capture model'. This suggests that cubosome encapsulation may not be an effective strategy for all antimicrobial compounds, paving the way for improved selection of nanocarriers for AMPs, and other antimicrobial compounds.

Overcoming Therapeutic Challenges of Antibiotic Delivery with Cubosome Lipid Nanocarriers.

Low discovery rates for new antibiotics, commercial disincentives to invest, and inappropriate use of existing drugs have created a perfect storm of antimicrobial resistance (AMR). This "silent pandemic" of AMR looms as an immense, global threat to human health. In tandem, many potential novel drug candidates are not progressed due to elevated hydrophobicity, which may result in poor intracellular internalization and undesirable serum protein binding. With a reducing arsenal of effective antibiotics, enabling technology platforms that improve the outcome of treatments, such as repurposing existing bioactive agents, is a prospective option. Nanocarrier (NC) mediated drug delivery is one avenue for amplifying the therapeutic outcome. Here, the performance of several antibiotic classes encapsulated within the lipid-based cubosomes is examined. The findings demonstrate that encapsulation affords significant improvements in drug concentration:inhibition outcomes and assists in other therapeutic challenges associated with internalization, enzyme degradation, and protein binding. We emphasize that a currently sidelined compound, novobiocin, became active and revealed a significant increase in inhibition against the pathogenic Gram-negative strain, Pseudomonas aeruginosa. Encapsulation affords co-delivery of multiple bioactives as a strategy for mitigating failure of monotherapies and tackling resistance. The rationale in optimized drug selection and nanocarrier choice is examined by transport modeling which agrees with experimental inhibition results. The results demonstrate that lipid nanocarrier encapsulation may alleviate a range of challenges faced by antibiotic therapies and increase the range of antibiotics available to treat bacterial infections.

A systematic study of the effect of lipid architecture on cytotoxicity and cellular uptake of cationic cubosomes.

HYPOTHESIS: Lipid nanoparticles containing a cationic lipid are increasingly used in drug and gene delivery as they can display improved cellular uptake, enhanced loading for anionic cargo such as siRNA and mRNA or exhibit additional functionality such as cytotoxicity against cancer cells. This research study tests the hypothesis that the molecular structure of the cationic lipid influences the structure of the lipid nanoparticle, the cellular uptake, and the resultant cytotoxicity. EXPERIMENTS: Three potentially cytotoxic cationic lipids, with systematic variations to the hydrophobic moiety, were designed and synthesised. All the three cationic lipids synthesised contain pharmacophores such as the bicyclic coumarin group (CCA12), the tricyclic etodolac moiety (ETD12), or the large pentacyclic triterpenoid "ursolic" group (U12) conjugated to a quaternary ammonium cationic lipid containing twin C12 chains. The cationic lipids were doped into monoolein cubosomes at a range of concentrations from 0.1 mol% to 5 mol% and the effect of the lipid molecular architecture on the cubosome phase behaviour was assessed using a combination of Small Angle X-Ray Scattering (SAXS), Dynamic Light Scattering (DLS), zeta-potential and cryo-Transmission Electron Microscopy (Cryo-TEM). The resulting cytotoxicity of these particles against a range of cancerous and non-cancerous cell-lines was assessed, along with their cellular uptake. FINDINGS: The molecular architecture of the cationic lipid was linked to the internal nanostructure of the resulting cationic cubosomes with a transition to more curved cubic and hexagonal phases generally observed. Cubosomes formed from the cationic lipid CCA12 were found to have improved cellular uptake and significantly higher cytotoxicity than the cationic lipids ETD12 and U12 against the gastric cancer cell-line (AGS) at lipid concentrations ≥ 75 µg/mL. CCA12 cationic cubosomes also displayed reasonable cytotoxicity against the prostate cancer PC-3 cell-line at lipid concentrations ≥ 100 µg/mL. In contrast, 2.5 mol% ETD12 and 2.5 mol% U12 cubosomes were generally non-toxic against both cancerous and non-cancerous cell lines over the entire concentration range tested. The molecular architecture of the cationic lipid was found to influence the cubosome phase behaviour, the cellular uptake and the toxicity although further studies are necessary to determine the exact relationship between structure and cellular uptake across a range of cell lines.

pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages.

mRNA lipid nanoparticle (LNP) technology presents enormous opportunities to prevent and treat various diseases. Here, we developed a novel series of LNPs containing ionizable amino-lipids showing a remarkable array of tunable and pH-sensitive lyotropic liquid crystalline mesophases including the inverse bicontinuous cubic and hexagonal phases characterized by high-throughput synchrotron radiation X-ray scattering. Furthermore, with an interest in developing mRNA therapeutics for lung macrophage targeting, we discovered that there is a strong correlation between the mesophase transition of the LNPs during acidification and the macrophage association/transfection efficiency of mRNAs. The slight molecular structural differences between the SM-102 and ALC-0315 ionizable lipids are linked to the LNP's ability to transform their internal structures from an amorphous state to the inverse micellar, hexagonal, and finally cubic structures during endosomal maturation. SM-102 LNPs showed exceptionally improved transfection efficiency due to their ability to form a cubic structure at a lower pH than the ALC-0315 analogues, which remained within the hexagonal structure, previously attributed to promoting endosomal escape of the ionizable LNPs. Overall, the new knowledge draws our attention to the important role of mesophase transition in endosomal escape, and the novel LNP libraries reported herein have broad prospects for advancing mRNA therapeutics.

Real-Time pH-Dependent Self-Assembly of Ionisable Lipids from COVID-19 Vaccines and In Situ Nucleic Acid Complexation.

Ionisable amino-lipid is a key component in lipid nanoparticles (LNPs), which plays a crucial role in the encapsulation of RNA molecules, allowing efficient cellular uptake and then releasing RNA from acidic endosomes. Herein, we present direct evidence for the remarkable structural transitions, with decreasing membrane curvature, including from inverse micellar, to inverse hexagonal, to two distinct inverse bicontinuous cubic, and finally to a lamellar phase for the two mainstream COVID-19 vaccine ionisable ALC-0315 and SM-102 lipids, occurring upon gradual acidification as encountered in endosomes. The millisecond kinetic growth of the inverse cubic and hexagonal structures and the evolution of the ordered structural formation upon ionisable lipid-RNA/DNA complexation are quantitatively revealed by in situ synchrotron radiation time-resolved small angle X-ray scattering coupled with rapid flow mixing. We found that the final self-assembled structural identity, and the formation kinetics, were controlled by the ionisable lipid molecular structure, acidic bulk environment, lipid compositions, and nucleic acid molecular structure/size. The implicated link between the inverse membrane curvature of LNP and LNP endosomal escape helps future optimisation of ionisable lipids and LNP engineering for RNA and gene delivery.

Prospective Subunit Nanovaccine against Mycobacterium tuberculosis Infection─Cubosome Lipid Nanocarriers of Cord Factor, Trehalose 6,6' Dimycolate.

An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.

Cubosome Lipid Nanocarriers As a Drug Delivery Vehicle for Intracellular Mycobacterium tuberculosis Infections.

Mycobacterium tuberculosis (MTB) causes the infectious disease tuberculosis (TB), responsible for more deaths than any other single infectious disease in history. Intracellular MTB are slow growing and difficult to target with traditional antitubercular drugs, leading to the emergence of multidrug resistance in TB infection, which is a major global public health issue. Recent advances in innovative lipid nanotechnologies for drug delivery have demonstrated promising outcomes for chronic infectious diseases but have not yet been tested as potential delivery systems for intracellular infections such as TB. The current study evaluates the potential of monoolein (MO)-based cationic cubosomes for the encapsulation and delivery of the first line antitubercular drug rifampicin (RIF) against an MTB-H37Ra in vitro culture model. In particular, we show that the use of cationic cubosomes as delivery vehicles reduced the minimum inhibitory concentration (MIC) of RIF by 2-fold against actively replicating MTB-H37Ra (compared to that of the free drug) and also shortened the lifecycle duration of axenic MTB-H37Ra from 5 to 3 days. The cubosome-mediated delivery was also found to be effective against intracellular MTB-H37Ra within THP-1 human macrophages, with a 2.8 log reduction in viability of the bacilli after 6 days incubation at the MIC. The killing time was also reduced from 8 to 6 days without distressing the host macrophages. Mechanistic studies on the uptake of RIF-loaded cationic cubosomes using total internal reflection fluorescence microscopy (TIRFM) demonstrated the capacity of these lipid particles to effectively target intracellular bacteria. Overall, these results demonstrate that cationic cubosomes are a potent delivery system for the antitubercular drug RIF for therapeutic management of TB.

An Engineered Nanosugar Enables Rapid and Sustained Glucose-Responsive Insulin Delivery in Diabetic Mice.

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are "nanosugars" bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL-1 ) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar.

A promising new oral delivery mode for insulin using lipid-filled enteric-coated capsules.

The treatment of diabetes requires daily administration of the peptide insulin via subcutaneous (SC) injection due to poor stability following oral administration. Enteric capsules, designed to protect against low pH conditions in the stomach by providing a polymeric coating which only breaks down in the small intestine, have failed to significantly increase oral bioavailability for insulin. In parallel, amphiphilic lipid mesophases are versatile carrier materials which can protect encapsulated proteins and peptides from undesirable enzymatic degradation. Here we show the combined delivery capacity of a hydrated bicontinuous cubic lipid mesophase embedded within an enteric capsule. Animal studies demonstrated that the lipid filled enteric capsules could deliver insulin with bioavailabilities (relative to SC injection) as high as 99 % and 150 % for fast and slow acting insulin, respectively. These results provide a promising starting point towards further trials to develop an alternative, non-invasive mode for the delivery of insulin.

Formation of particulate lipid lyotropic liquid crystalline nanocarriers using a microfluidic platform.

HYPOTHESIS: Non-lamellar lyotropic liquid crystal nanoparticles (LLCNPs) are gaining significant interest in the fields of drug delivery and nanomedicine. Traditional, top-down formulation strategies for LLCNPs are typically low-throughput, can lack controllability and reproducibility in the particle size distribution, and may be unsuitable for loading more fragile therapeutics. The development of a controllable, reproducible, scalable, and high-throughput strategy is urgently needed. EXPERIMENTS: Monoolein (MO)-based LLCNPs with various stabilizers (F127, F108, and Tween 80) and phytantriol (PT)-F127 cubosomes were produced at various flow conditions via a bottom-up method using a microfluidic platform. FINDINGS: This simple enabling strategy was used to formulate LLCNPs with lower polydispersity compared to the traditional top-down homogenization method. Significantly, particle size could be quantitatively controlled by varying the overall flow-rate; a scaling law was identified between nanoparticle mean size and the total flow rate (Q) of meansize∼Q-0.15 for MO cubosomes and meansize∼Q-0.19 for PT cubosomes (at a fixed flow rate ratio). Effective size control was achieved for a range of cubosome formulations involving different lipids and stabilizers. The formulation of stable, drug-loaded cubosomes with high encapsulation efficiency using this method was exemplified using calcein as a model drug. This work will further promote the utilisation of LLCNPs in nanomedicine and facilitate their clinical translation.

Enhancing the photoluminescence and cellular uptake of fluorescent carbon nanodots via cubosome lipid nanocarriers.

Carbon nanodots (C-dots) have attracted much attention for their use in the fields of bioimaging, drug delivery, and sensing due to their excellent fluorescent and photoluminescent properties, photostability, biocompatibility, and amenability to surface modification. Herein, we report a nanocomposite formulation of C-dots (<5 nm) encapsulated in lipid-based lyotropic liquid crystalline nanoparticles (∼250 nm) via either passive diffusion or electrostatic mechanisms. The physicochemical properties of the nanocomposite formulation including particle size, surface charge, internal cubic nanostructures, and pH-dependent fluorescent properties were characterised. Upon loading of C-dots into lipid nanoparticles, the highly ordered inverse bicontinuous cubic mesophase existed in the internal phase of the nanoparticles, demonstrated by synchrotron small angle X-ray scattering, molecular dynamic simulation and cryogenic transmission electron microscopy. The pH-dependent fluorescent property of the C-dots was modified via electrostatic interaction between the C-dots and cationic lipid nanoparticles, which further enhanced the brightness of C-dots through self-quenching prevention. The cytotoxicity and cellular uptake efficiency of the developed nanocomposites were also examined in an epithelial gastric adenocarcinoma cell line (AGS) and a macrophage cell line (stimulated THP-1). Compared to free C-dots, the uptake and cell imaging potential of the C-dot nanocomposites was significantly improved, by several orders of magnitude as demonstrated by cytoplasmic fluorescent intensities using confocal microscopy. Loading C-dots into mesoporous lipid nanocarriers presents a new way of modifying C-dot physicochemical and fluorescent properties, alternative to direct chemical surface modification, and advances the bioimaging potential of C-dots by enhancing cellular uptake efficiency and converging C-dot light emission.

Lipid membrane-mediated assembly of the functional amyloid-forming peptide Somatostatin-14.

Membrane-mediated assembly has been well characterised for toxic amyloid species such as the amyloid-ß peptide implicated in Alzheimer's disease. However, little is known about the membrane-mediated assembly of functional-amyloid forming peptides, recently identified as a natural storage state for neuropeptide hormones in vivo. Here, we study the aggregation of somatostatin-14 (SST-14) co-incubated with model lipid membranes. Atomic force microscopy (AFM) studies confirmed that nanofibrils formed in the presence of various lipid membranes display reduced fibrillogenesis and promote the formation of non-fibrillar oligomers. Both circular dichroism (CD) and intrinsic tryptophan fluorescence studies confirmed interaction between the peptide and the lipid bilayer; this interaction appears to drive changes in membrane-mediated aggregation kinetics. We show that both the surface charge of the membrane and chain packing drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of assembly. The similarities in the effect of the lipid membrane on aggregation of functional amyloids and the more well studied toxic amyloids suggest strong aggregation modifying lipid bilayer interactions are a ubiquitous feature of all amyloid fibrils and highlight the need for further investigation as to why this leads to toxicity in some systems and not others.

Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs.

Engineered nanoparticles for the encapsulation of bioactive agents hold promise to improve disease diagnosis, prevention and therapy. To advance this field and enable clinical translation, the rational design of nanoparticles with controlled functionalities and a robust understanding of nanoparticle-cell interactions in the complex biological milieu are of paramount importance. Herein, a simple platform obtained through the nanocomplexation of glycogen nanoparticles and albumin is introduced for the delivery of chemotherapeutics in complex multicellular 2D and 3D systems. We found that the dendrimer-like structure of aminated glycogen nanoparticles is key to controlling the multivalent coordination and phase separation of albumin molecules to form stable glycogen-albumin nanocomplexes. The pH-responsive glycogen scaffold conferred the nanocomplexes the ability to undergo partial endosomal escape in tumour, stromal and immune cells while albumin enabled nanocomplexes to cross endothelial cells and carry therapeutic agents. Limited interactions of nanocomplexes with T cells, B cells and natural killer cells derived from human blood were observed. The nanocomplexes can accommodate chemotherapeutic drugs and release them in multicellular 2D and 3D constructs. The drugs loaded on the nanocomplexes retained their cytotoxic activity, which is comparable with the activity of the free drugs. Cancer cells were found to be more sensitive to the drugs in the presence of stromal and immune cells. Penetration and cytotoxicity of the drug-loaded nanocomplexes in tumour mimicking tissues were validated using a 3D multicellular-collagen construct in a perfusion bioreactor. The results highlight a simple and potentially scalable strategy for engineering nanocomplexes made entirely of biological macromolecules with potential use for drug delivery.

Uptake Dynamics of Cubosome Nanocarriers at Bacterial Surfaces and the Routes for Cargo Internalization.

Antibiotic-resistant bacteria pose a significant threat to humanity. Gram-negative strains have demonstrated resistance to last resort antibiotics, partially due to their outer membrane, which hinders transport of antimicrobials into the bacterium. Nanocarrier (NC)-mediated drug delivery is one proposed strategy for combating this emerging issue. Here, the uptake of self-assembled lipid nanocarriers of cubic symmetry (cubosomes) into bacteria revealed fundamental differences in the uptake mechanism between Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the NCs adhere to the outer peptidoglycan layers and slowly internalize to the bacterium. For Gram-negative bacteria, the NCs interact in two stages, fusion with the outer lipid membrane and then diffusion through the inner wall. The self-assembled nature of the cubosomes imparts a unique ability to transfer payloads via membrane fusion. Remarkably, the fusion uptake mechanism allowed rapid NC internalization by the Gram-negative bacteria, overcoming the outer membrane responsible for their heightened resilience. Here this is demonstrated by the marked reduction in the minimal inhibition concentration required for antibiotics against a pathogenic strain of Gram-negative bacteria, Escherichia coli. These results provide mechanistic insight for the development of lipid NCs as a new tool to combat bacteria.

Delivery of antimicrobial peptides to model membranes by cubosome nanocarriers.

Antimicrobial peptides (AMPs), which typically disrupt the bacterial wall prompting leakage or lysis of the cell, form a growing contingent in the arsenal against antibiotic resistant bacteria. The effectiveness of AMPs is, however, hampered by their low solubility, general chemical and physical instability, and short half-life in vivo. Lipid nanocarriers such as cubosomes are effective at encapsulating and protecting proteins while simultaneously showing promise in delivery applications. Here, the efficacy of cubosome mediated delivery of AMPs is evaluated by the in-situ surface characterization of model membranes with varying composition. The cubosomes were observed to initially fuse with the membranes, with subsequent membrane disruption observed after approximately 20 - 60 min. The time for the disruption was sensitive to the charge of the cubosome as well as the composition of the bilayer. More physiologically relevant bilayers including lipids with phospho-(1'-rac-glycerol) (PG) or phosphoethanolamine (PE) headgroups were more vulnerable than those of neat phosphocholine (PC). Notably, disruption to the bilayer occurred an order of magnitude faster for encapsulated AMP compared to free AMP.

Reduction of enzymatic degradation of insulin via encapsulation in a lipidic bicontinuous cubic phase.

Oral delivery of the protein drug insulin is not currently possible due to rapid degradation of the secondary structure in low pH conditions in the stomach and under the influence of digestive enzymes in the gastrointestinal tract. Effective oral delivery of insulin and other protein- or peptide-based drugs will, therefore, require encapsulation in a material or nanoparticle. Herein we investigate the ability of the lipid bicontinuous cubic phase formed by two lipids, monoolein (MO) and phytantriol (PT), to protect encapsulated insulin from degradation by the enzyme chymotrypsin, typically found in the small intestine. High encapsulation efficiency (>80%) was achieved in both lipid cubic phases with retention of the underlying cubic nanostructure. Release of insulin from the cubic matrix was shown to be diffusion-controlled; the release rate was dependent on the cubic nanostructure and consistent with measured diffusion coefficients for encapsulated insulin. Encapsulation was shown to significantly retard enzymatic degradation relative to that in water, with the protective effect lasting up to 2 h, exemplifying the potential of these materials to protect the encapsulated protein payload during oral delivery.

Encapsulated Nanodroplets for Enhanced Fluorescence Detection by Nano-Extraction.

Enhancement of the detection signal of fluorescence microscopy in highly diluted solutions is of great importance in chemical analysis, sensing, and bioassay applications. Surface nanodroplets with atto- to femto-liter volumes are promising tools for sensitive online detection by integrating their extremely efficient nano-extraction and optical advantages. In this paper, the development of novel basic units of nanodroplets-in-a-microdroplet by simple solvent exchange is reported. The encapsulated nanodroplets are applied for ultrasensitive and online detection in fluorescence imaging. The biphasic nature of the droplet composite enables simultaneous extraction and enrichment of both hydrophobic and hydrophilic compounds. Furthermore, the desirable lensing effect of the curved surface of the nanodroplets enhances the collection of light emitted from the fluorophore extracted in the droplets by ≈60-fold, allowing sensitive and quantitative analysis of the fluorophore using fluorescence microscopy. The results highlight the potential of encapsulated nanodroplets as a simple and innovative method of signal enhancement in chemical analysis. By integrating selective concentration, extraction, and sensitive detection, the encapsulated nanodroplets reported here may have broad applications in many chemical and biological matrices.

Accelerated Formation of H2 Nanobubbles from a Surface Nanodroplet Reaction.

The compartmentalization of chemical reactions within droplets has advantages in low costs, reduced consumption of reagents, and increased throughput. Reactions in small droplets have also been shown to greatly accelerate the rate of many chemical reactions. The accelerated growth rate of nanobubbles from nanodroplet reactions is demonstrated in this work. The gaseous products from the reaction at the nanodroplet surface promoted nucleation of hydrogen nanobubbles within multiple organic liquid nanodroplets. The nanobubbles were confined within the droplets and selectively grew and collapsed at the droplet perimeter, as visualized by microscopy with high spatial and temporal resolutions. The growth rate of the bubbles was significantly accelerated within small droplets and scaled inversely with droplet radius. The acceleration was attributed to confinement from the droplet volume and effect from the surface area on the interfacial chemical reaction for gas production. The results of this study provide further understanding for applications in droplet enhanced production of nanobubbles and the on-demand liberation of hydrogen.