Preformed Vesicles Research Articles

Characterization and Optimization of Vesicle Properties in bioPISA: from Size Distribution to Post-Assembly Loading.

This study investigates the formation and properties of vesicles produced via biocatalytic Polymerization-Induced Self-Assembly (bioPISA) as artificial cells. Methods for achieving size uniformity, including gentle centrifugation and sucrose gradient centrifugation, are explored, and the effects of stirring speed on vesicle morphology is investigated. The internal structure of the vesicles, characterized by a polymer-rich matrix, is analyzed using fluorescence correlation spectroscopy (FCS). Additionally, the feasibility of loading macromolecules into pre-formed vesicles is demonstrated using electroporation, and a fluorescent protein as well as enzymes for a cascade reaction were sucesfully incorporated into the fully assembled polymersomes. These findings provide a foundation for developing enzyme-synthesized polymeric vesicles with controlled morphologies for various applications, e.g., in synthetic biology.

Peptide-Induced Division of Polymersomes for Biomimetic Compartmentalization.

Polymersomes are synthetic vesicles that mimic the architecture of cellular compartments such as the cell membrane and organelles. These biomimetic compartments facilitate the creation of cell-like chemical systems, including microreactors and synthetic organelles. However, the construction of hierarchical multi-compartment systems remains challenging and typically requires the encapsulation of pre-formed vesicles within a host compartment. Here, we report the formation of multicompartment polymersomes with a vesicle-in-vesicle architecture achieved through self-division induced by short peptides incorporated into the vesicle membrane. A phenylalanine-phenylalanine-methionine (FFM) tripeptide was designed and encapsulated into the polymersome via microfluidics. We demonstrate that vesicle self-division occurs due to peptide incorporation into the membrane in response to pH changes. This self-division creates internal vesicles capable of colocalizing enzymes. The hybrid polymer-peptide system described here provides a straightforward method for developing subcompartmentalized systems, paving the way for engineering microreactors with life-like properties.

MRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles.

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.

Pentacyclic triterpenes modulate liposome membrane fluidity and permeability depending on membrane cholesterol content

Since the membrane-related processes represent an integral part of the biological activities of drugs, their effect on the membrane dynamics is actually considered. In this study, we investigated the effect of pentacyclic triterpenes (TTPs), oleanolic acid (OA) and erythrodiol (ER), on the fluidity and permeability of liposomes membranes differing by their cholesterol content. All liposomes were prepared by reverse phase evaporation technique (REV). Spin-labeled liposomes exposed or not to TTPs were used for fluidity studies by using 5- and 16-doxyl stearic acids (DSA). TTPs-loaded liposomes (phospholipid:cholesterol of 1:1), and preformed vesicles exposed to TTPs were used for permeability studies by monitoring the release of sulforhodamine B (SRB) at 37 °C. The apparent release constants of SRB were determined by Higuchi model based on a biphasic curve shape (0–10 h; 10–48 h). TTPs-loaded liposomes were characterized for their size and homogeneity. Results showed that ER increased the membrane fluidity at the upper region of the membrane while the both TTPs produced a condensing effect at the deeper region of the membrane. The membrane composition was a critical parameter modulating the effect of TTPs on the membrane permeability. Also, this study consolidated the fact that a fluidizing membrane agent is not necessarily a permeabilizing-membrane compound.

Extracellular vesicles as antigen carriers for novel vaccination avenues

Antigen delivery has always been a challenge in scientific practice of vaccine formulation. Yet, mammalian extracellular vesicles (EVs) or bacterial membrane vesicles (MVs) provide an innovative avenue for safe and effective delivery of antigenic material. They include intrinsically loaded antigens from EV-secreting cells or extrinsically loaded antigens onto pre-formed vesicles. Interestingly, many studies shed light on potential novel anti-cancer vaccination immunotherapy for therapeutic applications from mammalian cell host-derived EVs, as well as conventional vaccination for prophylactic applications using bacterial cell-derived MVs against infectious diseases. Here, we discuss the rationale, status quo and potential for both vaccine applications using EVs.

Hybrid Vesicular Nanosystems Based on Lipids and Polymers Applied in Therapy, Theranostics, and Cosmetics.

Nanotechnology has made great contributions in the development of materials with potential application in different areas, especially in the pharmaceutical sector, where nano-systems are being intensely studied for controlled drug release. These innovative systems are composed of structures such as nanoparticles, nanoemulsions, and cyclodextrins, with the aim of promoting enhanced bioavailability of bioactive molecules. Among these nanocarriers, vesicles such as liposomes and polymersomes are considered to be promising alternatives in delivering hydrophilic and lipophilic drugs. They have different classifications according to their composition, among which are hybrid vesicles, which unlike liposomes are composed of both lipids and polymers. These vesicular systems stand out for combining the advantages of both components, overcoming the limitations of traditional systems imposed by low stability and premature release of the encapsulated active substance. The polymers applied in hybrid vesicles can make up the membrane structure itself or be employed to coat preformed vesicles. Due to the relevance of these systems, this work covers their characteristics and summarizes recent articles about them in the literature.

Design and Applications of a Fluorescent Labeling Technique for Lipid and Surfactant Preformed Vesicles.

Amphiphilic molecules such as surfactants, lipids, and block copolymers can be assembled into bilayers and form vesicles. Fluorescent membrane labeling methods require the use of dye molecules that can be inserted into the bilayers at different stages of synthesis. To our knowledge, there is no generalized method for labeling preformed vesicles. Herein, we develop a versatile protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. On the basis of the lipophilic carbocyanine green dye PKH67, the methodology is assessed on zwitterionic phosphatidylcholine vesicles. To demonstrate its versatility, it is applied to dispersions of anionic or cationic vesicles, such as a drug administrated to premature infants with respiratory distress syndrome, or a vesicle formulation used as a fabric softener for home care applications. By means of fluorescence microscopy, we then visualize the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactants adsorbed on a cotton fabric. These results highlight the advantages of a membrane labeling technique that is simple and applicable to a large number of soft matter systems.

Towards unidirectional reconstitution of membrane proteins into liposomes

Functional studies of membrane proteins (MP) in their native environment (the cellular membrane) is challenging due to the complexity of the native membranes and measurement interferences from other membrane constituents or reactions. Therefore, MPs are often purified and integrated into systems which mimic their natural environment in a membrane as e.g. liposomes or nanodiscs, a process called reconstitution, allowing to investigate its function and structural aspects without any disturbing background. The most important problem during MP-reconstitution is the often random orientation of the MP in the liposomal membrane after reconstitution. For functional studies of the MP of interest and quantitative analysis of its properties, unidirectional orientation in the liposomal membrane is required. Previous work of other groups, where MPs have been reconstituted and studied, did not include a final and universal approach (Rigaud JL, Levy D. (2003) Methods Enzymol. 372:65-86.; Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96.) and procedures have to be individually optimized for an enrichment of enzyme orientation. In most cases, however, orientation cannot be influenced and is thought to depend on the three-dimensional structure of the protein (Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96; Nordlund G et al. (2014) Nat. Commun. 5:4303.). We are currently developing and establishing a universal method to force unidirectional reconstitution of MPs by the aid of a molecular unit that can be attached to every protein. Our method is based on the observation that the F1F0 ATP synthase with its large hydrophilic head piece (F1 part) orients highly uniformly when embedded in preformed vesicles. We try to mimic this situation with a bioengineering approach that should be applicable to a wide variety of proteins. Once such a method is fully established, the interplay of two or more membrane proteins can be investigated more quantitatively.

New Insights into the Growth and Transformation of Vesicles: A Free-Flow Electrophoresis Study.

The spontaneous formation of lipid vesicles, in particular fatty acid vesicles, is considered an important physical process at the roots of cellular life. It has been demonstrated previously that the addition of fatty acid micelles to preformed vesicles induces vesicle self-reproduction by a growth-division mechanism. Despite multiple experimental efforts, it remains unresolved how vesicles rearrange upon the addition of fresh membrane-forming compounds, and whether solutes that are initially encapsulated inside the mother vesicles are evenly redistributed among the daughter ones. Here we investigate the growth-division of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles, which, following the addition of oleate micelles, form mixed oleate/POPC vesicles. Our approach is based on free-flow electrophoresis (FFE) and cryogenic transmission electronmicroscopy (cryo-TEM). Two new features emerge from this study. FFE analysis unexpectedly reveals that the uptake of oleate micelles by POPC vesicles follows two different pathways depending on the micelles/vesicles ratio. At low oleate molar fractions (<0.35), plain incorporation of oleate into pre-existing POPC vesicles is our dominant observation. In contrast, oleate-rich and oleate-poor daughter vesicles are generated from parent POPC vesicles when the oleate molar fraction exceeds 0.35. Cryo-TEM reveals that when ferritin-filled vesicles grow and divide, some vesicles contain ferritin at increased concentrations, others are empty. Intriguingly, in some cases, ferritin appears to be highly concentrated inside the vesicles. These observations imply a specific redistribution (partitioning) of encapsulated solutes among nascent vesicles during the growth-division steps. We have interpreted our observations by assuming that freshly added oleate molecules are taken-up preferentially (cooperatively) by oleate-rich membrane regions that form spontaneously in POPC/oleate vesicles when a certain threshold (oleate molar fraction ca. 0.35) is surpassed. The proposed cooperative mechanism could be based on differential microscopic constants for oleate/oleic acid dynamics in oleate-rich and oleate-poor membrane regions, which eventually generate populations of oleate-rich and oleate-poor vesicles.

Characterization of iNOS+ Neutrophil-like ring cell in tumor-bearing mice

BackgroundMyeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset.MethodsSingle-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro.ResultsThe results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells.ConclusionsIn this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Copper-free click chemistry on polymersomes: pre- vs. post-self-assembly functionalisation

The optimal accessibility of functional groups on polymeric nanosized vesicles was investigated with copper-free clickable probes as a model system. Cu-free clickable polymersomes were developed either through co-assembly of end group modified amphiphilic block copolymers or by introduction of the reactive moieties on preformed vesicles. For the co-assembly approach, the highest degree of availability was obtained for the most hydrophilic functional group, whereas hydrophobic species were unable to react as efficiently since they were seemingly buried in the membrane. Post-self-assembly introduction led to good results for all three examined moieties whereby surface saturation was reached above a certain percentage of immobilised probes. Finally, we demonstrated that protrusion of functional entities from the membrane corona via a longer hydrophilic segment of the block-copolymer significantly enhances the accessibility.

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: Distearoylphosphatidylethanolamine (DSPE)-PEG2000 was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG1100 was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG2000 or sterol-PEG1100 into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG1100 in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.

Orosensory Stimulation Effects on Human Saliva Proteome

Saliva flow induced by 6-gingerol (pungent), hydroxy-α/β-sanshools (tingling), and citric acid (sour) was measured, and the time-dependent changes in the whole saliva proteome were analyzed by means of 2D-PAGE, followed by tryptic in-gel digestion and MALDI-TOF-MS peptide mass fingerprint analysis. The proteins showing significantly decreased abundance after oral 6-gingerol stimulation were identified as glutathione S-transferase P, the heat shock protein β-1, the heat shock 70 kDa protein 1, annexin A1, and cytoplasmic β-actin, whereas prolactin inducible proteins (PIP), short palate, lung and nasal epithelium carcinoma-associated protein 2 (SPLUNC2), zinc-α-2-glycoproteins (Zn-α-GP), and carbonic anhydrase VI (CAVI) were found with increased abundance. As the effects of this study were observed instantaneously upon stimulation, any proteome modulation is very likely to result from the release of proteins from preformed vesicles and not from de novo synthesis. The elevated levels of SPLUNC2, Zn-α-GP, and CAVI might be interpreted to trigger innate protective mechanisms in mucosal immunity and in nonimmune mucosal defense and might play an important role during the initial stage of inflammation.

The World Constructed by Self-organization of Some Amphiphils-with a Focus on Vesicle Formation-

The world constructed by self-organization of some amphiphils was discussed on the basis of micelle formation, vesicle formation, and oriented-nano-wire formation. First, the micelle formation of a both water- and oil- soluble surfactant, Aerosol OT, was discussed. Solution states of micelles and monomer were discussed on the basis of thermodinamic and NMR spectroscopic analyses of micelle formation. Next, micelle-vesicle transition was discussed. It was proposed that the phospholipid LUV formation by removing detergents and destruction by adding detergents occurred via 4 stages. The 4 stage model instead of the 3 stage model could not only elucidate the complicated phenomena observed during micelle-vesicle transition, but predicted the size and properties of the vesicles formed by detergent removal from mixed micelles. Next, the vesicle formation of a fatty acid with a single hydrophobic chain different from phospholipid, which has two hydorophobic chains, was discussed. The vesicle formation was strongly affected by the presence of preformed vesicles and the size was biased on the preformed vesicles. It was shown there exist two pass ways in the process of micelle-vesicle transition by pH jump. One is fission of the preformed vesicles after transfer of monomers from newly added oleate micelles and the other is transition from the mixed micelles after partial solubilization by the oreate micelles. Then, the vesicle formation of HCO-10, which has 3 hydrophobic chains, the mixed vesicle formation of phosphatidylethanolamine and lysophosphtidylcholine, which can not form vesicles, and the phospholipid vesicle formation and destruction by removing and adding PEG-lipid, were discussed. Lastly, oriented nano wire formation of mulamyldipeptid-conjugated lipids with ca 5 nm of diameter was discussed.

Hydrophobic Gold Nanoparticle Self-Assembly with Phosphatidylcholine Lipid: Membrane-Loaded and Janus Vesicles

Hybrids of hydrophobic sub-2-nm-diameter dodecanethiol-coated Au nanoparticles and phosphatidylcholine (PC) lipid vesicles made by extrusion were examined by cryogenic transmission electron microscopy (cryoTEM). The nanoparticles loaded the vesicles as a dense monolayer in the hydrophobic core of the lipid bilayer, without disrupting their structure. Nanoparticle-vesicle hybrids could also be made by a dialysis process, mixing preformed vesicles with detergent-stabilized nanoparticles, but this approach led to vesicles only partially loaded with nanoparticles that segregated into hemispherical domains, forming a Janus vesicle-nanoparticle hybrid structure.

Bone Marrow-Targeted Liposomal Carriers: A Feasibility Study in Nonhuman Primates

Recently, we described a novel surface-modified lipid vesicle formulation (liposome) that had very high targeting to bone marrow in normal rabbits. Because the bone marrow is the site of hematopoiesis, bone marrow-targeted drug-delivery systems have many potential applications. In this study we investigated whether these bone marrow-targeted vesicles are also similarly effective for bone marrow targeting in rhesus monkeys, a primate animal model that is more relevant to humans. The preformed vesicles encapsulating 30 mM glutathione were labeled with technetium-99m ((99m)Tc) for scintigraphic imaging. The vesicles were 216 +/- 21 nm in diameter with a negative surface charge composed of DPPC, cholesterol, anionic amphiphile and poly(ethylene glycol)-DSPE (1:1:0.2:0.013 molar ratio). The whole-body images of rhesus monkeys receiving intravenous (99m)Tc vesicles revealed high uptake of the (99m)Tc vesicles in bone marrow. Based on image analysis, we estimated that approximately 70% of the injected dose of the (99m)Tc vesicles was taken up by the bone marrow. This finding increases the feasibility of using this bone marrow-specific drug-delivery system for clinical applications.

Rational design and immunogenicity of liposome-based diepitope constructs: Application to synthetic oligosaccharides mimicking the Shigella flexneri 2a O-antigen

We have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307–319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam 3CAG, i.e. a TLR2 ligand known for its adjuvant properties, anchored in preformed vesicles. The synthetic construct containing a pentadecasaccharide corresponding to three O-antigen repeating units triggered T-dependent anti-oligosaccharide and anti- S. flexneri 2a LPS antibody responses when administered i.m. to BALB/c mice. Moreover, the long-lasting anti-LPS antibody response afforded protection against a S. flexneri 2a challenge. These results show that liposome diepitope constructs could be attractive alternatives in the development of synthetic carbohydrate-based vaccines.

Application of Mean Field Boundary Potentials in Simulations of Lipid Vesicles

A method is presented to enhance the efficiency of simulations of lipid vesicles. The method increases computational speed by eliminating water molecules that either surround the vesicle or reside in the interior of the vesicle, without altering the properties of the water at the membrane interface. Specifically, mean field force approximation (MFFA) boundary potentials are used to replace both the internal and external excess bulk solvent. In addition to reducing the cost of simulating preformed vesicles, the molding effect of the boundary potentials also enhances the formation and equilibration of vesicles from random solutions of lipid in water. Vesicles with diameters in the range from 20 to 60 nm were obtained on a nanosecond time scale, without any noticeable effect of the boundary potentials on their structure.

Morphological consequences of metal ion–peptide vesicle interaction

The triskelion peptide conjugate 1, having a Trp–Trp dipeptide unit on the three arms, was synthesized and studied for the interaction of peptide-based soft structures with metal ions, by fluorescence and microscopic analyses. We observed that fluorescence was significantly quenched upon addition of Cu(II) metal ions, whereas the addition of other metal ions also caused moderate to insignificant changes in the fluorescence emission, suggesting specificity of this triskelion peptide 1 for Cu(II) ions. The addition of Cu(II) and other metal ions also altered the morphology of preformed vesicles obtained from triskelion peptide 1 in a concentration-dependent fashion, as observed from microscopic analysis. Such metal-responsive soft structures may find potential use as novel materials for delivery and sensing applications.

Mechanisms Contributing to Tonic Release at the Cone Photoreceptor Ribbon Synapse

Time-resolved capacitance measurements in combination with fluorescence measurements of internal calcium suggested three kinetic components of release in acutely isolated cone photoreceptors of the tiger salamander. A 45-fF releasable pool, corresponding to about 1,000 vesicles, was identified. This pool could be depleted with a time constant of a few hundred milliseconds and its recovery from depletion was quite rapid (tau approximately 1 s). The fusion of vesicles in this pool was blocked by low-millimolar EGTA. Endocytosis was sufficiently slow that it is likely that refilling of the releasable pool occurred from preformed vesicles. A second, slower component of release (tau(depletion) approximately 3 s) was identified that was approximately twice the size of the releasable pool. This pool may serve as a first reserve pool that replenishes the releasable pool. Computer simulations indicate that the properties of the releasable and first reserve pools are sufficient to maintain synaptic signaling for several seconds in the face of near-maximal stimulations and in the absence of other sources of vesicles. Along with lower rates of depletion, additional mechanisms, such as replenishment from distal reserve pools and the fast recycling of vesicles, may further contribute to the maintenance of graded, tonic release from cone photoreceptors.