Triplet-Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC.

Triplet-triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet-triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

Antifungal Activity and Molecular Mechanisms of Copper Nanoforms against Colletotrichum gloeosporioides.

In this work, we have synthesized copper nanoforms (Cu NFs) using ascorbic acid as a reducing agent and polyvinylpyrrolidone as a stabilizer. Elemental characterization using EDS has shown the nanostructure to be of high purity and compare well with commercially sourced nanoforms. SEM images of both Cu NFs show some agglomeration. The in-house NFs had a better even distribution and size of the nanostructures. The XRD peaks represented a face-centered cubic structure of Cu2O. The commercially sourced Cu NFs were found to be a mixture of Cu and Cu2O. Both forms had a crystalline structure. Using these two types of Cu NFs, an antimicrobial study against Colletotrichum gloeosporioides, a devastating plant pathogen, showed the in-house Cu NFs to be most effective at inhibiting growth of the pathogen. Interestingly, at low concentrations, both Cu NFs increased fungal growth, although the mycelia appeared thin and less dense than in the control. SEM macrographs showed that the in-house Cu NFs inhibited the fungus by flattening the mycelia and busting some of them. In contrast, the mycelia were short and appeared clustered when exposed to commercial Cu NFs. The difference in effect was related to the size and/or oxidation state of the Cu NFs. Furthermore, the fungus produced a defense mechanism in response to the NFs. The fungus produced melanin, with the degree of melanization directly corresponding to the concentration of the Cu NFs. Localization of aggregated Cu NFs could be clearly observed outside of the model membranes. The large agglomerates may only contribute indirectly by a hit-and-bounce-off effect, while small structures may adhere to the membrane surface and/or internalize. Spatio-temporal membrane dynamics were captured in real time. The dominant dynamics culminated into large fluctuations. Some of the large fluctuations resulted in vesicular transformation. The major transformation was exo-bud/exo-cytosis, which may be a way to excrete the foreign object (Cu NFs).

Formation of Giant Unilamellar Vesicles Assisted by Fluorinated Nanoparticles.

In the quest to produce artificial cells, one key challenge that remains to be solved is the recreation of a complex cellular membrane. Among the existing models, giant unilamellar vesicles (GUVs) are particularly interesting due to their intrinsic compartmentalisation ability and their resemblance in size and shape to eukaryotic cells. Many techniques have been developed to produce GUVs all having inherent advantages and disadvantages. Here, the authors show that fluorinated silica nanoparticles (FNPs) used to form Pickering emulsions in a fluorinated oil can destabilise lipid nanosystems to template the formation of GUVs. This technique enables GUV production across a broad spectrum of buffer conditions, while preventing the leakage of the encapsulated components into the oil phase. Furthermore, a simple centrifugation process is sufficient for the release of the emulsion-trapped GUVs, bypassing the need to use emulsion-destabilising chemicals. With fluorescent FNPs and transmission electron microscopy, the authors confirm that FNPs are efficiently removed, producing contaminant-free GUVs. Further experiments assessing the lateral diffusion of lipids and unilamellarity of the GUVs demonstrate that they are comparable to GUVs produced via electroformation. Finally, the ability of incorporating transmembrane proteins is demonstrated, highlighting the potential of this method for the production of GUVs for artificial cell applications.

Cheap portable electroformed giant unilamellar vesicles preparation kit.

The membrane of a cell separates the internal and external media of the cell and contributes to a variety of important processes, including gradient maintenance and signal transduction. Synthetic lipid-made vesicles are commonly utilized as cell membrane model systems. These could be liposomes or giant unilamellar vesicles (GUVs) in most cases. Liposomes are typically less than 0.5 microns in size, limiting their use for most microscopy experiments. GUVs are a form of liposomes that ranges in size from 5 to 200 microns and are ideal for examining complex phase behaviors of biomembranes using the classical optical setting. This study details the step-by-step development of a portable, light and low-cost kit for generating GUVs by electroformation. Our kit contains an in-built electronic circuitry, and the GUV generation setup, consisting of 3 ITO-coated glasses with heating electrode connections. Approximately 600 µl of GUVs can be produced in one experiment, while the amount could be increased by changing the dimensions of the GUV generation setup. Finally, the originality of the study comes from the fact that many users from different fields unfamiliar with electronics can use our home-built cost-effective approach instead of their expensive commercial counterparts.

Screening of Protein-Based Ultrasmall Nanozymes for Building Cell-Mimicking Catalytic Vesicles.

Enzymes are an important component for bottom-up building of synthetic/artificial cells. Nanozymes are nanomaterials with intrinsic enzyme-like properties, however, the construction of synthetic cells using nanozymes is difficult owing to their high surface energy or large size. Herein, the authors show a protein-based general platform that biomimetically integrates various ultrasmall metal nanozymes into protein shells. Specifically, eight metal-based ultrasmall nano-particles/clusters are in situ incorporated into ferritin nanocages that are self-assembled by 24 subunits of ferritin heavy chain. As a nanozyme generator, such a platform is suitable for screening the desired enzyme-like activities, including peroxidase (POD), oxidase (OXD), catalase (CAT) and superoxide dismutase (SOD). After screening, it is found that Ru intrinsically possesses the highest POD-like and CAT-like activities, while Mn and Pt show the highest OXD-like and SOD-like activities, respectively. Additionally, the inducers/inhibitors of various nanozymes are screened from more than 50 compounds to improve or inhibit their enzyme-like activities. Based on the screened nanozymes and their inhibitors, a proof-of-conceptually constructs cell-mimicking catalytic vesicles to mimic or modulate the events of redox homeostasis in living cells. This study offers a type of artificial metalloenzyme based on nanotechnology and shows a choice for bottom-up enzyme-based synthetic cell systems in a fully synthetic manner.

Super-Resolution Imaging of Highly Curved Membrane Structures in Giant Vesicles Encapsulating Molecular Condensates.

Molecular crowding is an inherent feature of cell interiors. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (poly(ethylene glycol) and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs exhibit highly curved structures like nanotubes. Upon liquid-liquid phase separation their membrane deforms into apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes, and kinks, have dimensions below optical resolution. Here, these are studied with super-resolution stimulated emission depletion (STED) microscopy facilitated by immobilization in a microfluidic device. The cylindrical nature of the nanotubes based on the superior resolution of STED and automated data analysis is demonstrated. The deduced membrane spontaneous curvature is in excellent agreement with theoretical predictions. Furthermore, the membrane kink-like structure is resolved as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle, which describes the wettability contrast of the encapsulated phases to the membrane. Resolving these highly curved membrane structures with STED imaging provides important insights in the membrane properties and interactions underlying cellular activities.

Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis.

Liposomes and giant unilamellar vesicles (GUVs) in particular are excellent compartments for constructing artificial cells. Traditionally, their use requires bench-top vesicle growth, followed by experimentation under a microscope. Such steps are time-consuming and can lead to loss of vesicles when they are transferred to an observation chamber. To overcome these issues, we present an integrated microfluidic chip which combines GUV formation, trapping, and multiple separate experiments in the same device. First, we optimized the buffer conditions to maximize both the yield and the subsequent trapping of the vesicles in micro-posts. Captured GUVs were monodisperse with specific size of 18 ± 4 µm in diameter. Next, we introduce a two-layer design with integrated valves which allows fast solution exchange in less than 20 s and on separate sub-populations of the trapped vesicles. We demonstrate that multiple experiments can be performed in a single chip with both membrane transport and permeabilization assays. In conclusion, we have developed a versatile all-in-one microfluidic chip with capabilities to produce and perform multiple experiments on a single batch of vesicles using low sample volumes. We expect this device will be highly advantageous for bottom-up synthetic biology where rapid encapsulation and visualization is required for enzymatic reactions.

One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells.

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil-surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Deformation of giant unilamellar vesicles under osmotic stress.

Biological membrane plays an important role in maintaining an osmotic equilibrium between the cytoplasm and the extracellular solution of cells. Here, the giant unilamellar vesicles (GUVs) as cell models were used to investigate the effect of osmotic stress on phospholipid membranes. The deformation of GUVs, including inward budding and outward budding, was systematically investigated by the osmotic press from glucose, sucrose, LiCl, and KCl solutions. The permeability (P) of DMPC, DMPC/10 mol% Chol GUVs, DMPC/25 mol% Chol GUVs, and DMPC/40 mol% Chol GUVs in glucose, sucrose, LiCl, and KCl solutions were all obtained. The P value decreases with the addition of more cholesterol in the bilayer. The monovalent cations caused higher permeability of lipid bilayer membranes due to their combination with phospholipids. The molar flux of water (J) value was found to be the key factor for determining the deformation state from mainly inward budding to mainly outward budding. The findings in this paper may help us to understand cell transformation triggered with osmotic stress.