Small Unilamellar Vesicles Research Articles

Mechanistic Dissection of Cu(I)‐translocating P‐type ATPase Pumps by I n‐vitro Real‐Time Transport in Artificial Lipid Bilayer Vesicles

Copper is an essential element in biological systems where it plays key roles as a co-factor in diverse cuproenzymes and in signaling processes. Cu(I) transporting P-type ATPases (CopA) are primary active transmembrane transporter proteins that utilize the energy of ATP hydrolysis to catalyze the extrusion of Cu(I) ions across cell membranes. Their activity plays a gatekeeper role in controlling intracellular Cu(I) levels and homeostasis, a key process for survival in all kingdoms of life. In humans, mutations in these Cu(I) pumps lead to the Menkes syndrome and the Wilson's disease. In pathogenic bacteria CopA proteins can act as virulence factors to overcome the Cu(I) stress generated by the host defense system, thereby representing a potential therapeutic target against pathogens causing various fatal diseases. Even though the structure and function of these transporters has been established, the overall mechanism for substrate translocation, their transport kinetic parameters and electrogenicity remain elusive. In this work we developed a novel coupled experimental platform, centered on multiple fluorescence reporter probes that permits to study substrate, the transport mechanism and translocation kinetics in real-time in Cu(I)-pumps reconstituted in a native-like environment. We reconstituted the model Cu(I)-pump from E. coli (EcCopA) and its inactive mutants in artificial small unilamellar vesicles (proteoliposomes). We quantitatively characterized the chemistry underlying substrate transport, putative counterions and charge translocation by encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, Pyranine and Oxonol VI) responsive to diverse stimuli (Cu(I) substrate, pH and transmembrane potential). The study established that Cu(I) pumps are primary-active uniporters and electrogenic pumps that build a transmembrane potential by translocating one positive charge (Cu(I)) per ATP hydrolysis cycle without coupling it to counterion transport. This analysis further reveals mechanistic differences in substrate translocation by CopA proteins in comparison to other characterized P-type ATPase classes. Moreover, the developed approach can be extended as a general biophysical tool to investigate other P-type ATPase pumps in which the mechanism of substrate translocation has not yet been established.

Chemical and morphological effects of the contraceptive hormone 17 α-ethynylestradiol on fluid lipid membranes

The lack of studies involving the effects in human health associated with the chronic ingestion of pollutants lead to the path of investigating the action of these compounds in cell membrane models. We demonstrated the interaction (causes and consequences) of the hormone 17 α-ethinylestradiol (EE2) with lipid monolayers (prepared as Langmuir films) and bilayers prepared as small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs). Both fluidity and majority chemical composition of real plasma cell membrane were guaranteed using the phospholipid 1-palmitoil-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Surface pressure-mean molecular area (π-A) isotherms and PM-IRRAS measurements highlighted the strong interaction of EE2 with POPC monolayers, leading the hormone to remain at the air/water interface and promoting its penetration into the phospholipid hydrophobic chains. In the case of bilayers, the entrance of the hormone inside the SUV is likely facilitated by their high curvature. In GUVs, EE2 was responsible for changes in the spherical shape, forming structures like buds and lipid protrusions. The set of results indicates the strong effects of EE2 on fluid membranes, which is an important feature to predict its damage in human cells.

The effect of polydispersity, shape fluctuations and curvature on small unilamellar vesicle small-angle X-ray scattering curves.

Small unilamellar vesicles (20-100 nm diameter) are model systems for strongly curved lipid membranes, in particular for cell organelles. Routinely, small-angle X-ray scattering (SAXS) is employed to study their size and electron-density profile (EDP). Current SAXS analysis of small unilamellar vesicles (SUVs) often employs a factorization into the structure factor (vesicle shape) and the form factor (lipid bilayer electron-density profile) and invokes additional idealizations: (i) an effective polydispersity distribution of vesicle radii, (ii) a spherical vesicle shape and (iii) an approximate account of membrane asymmetry, a feature particularly relevant for strongly curved membranes. These idealizations do not account for thermal shape fluctuations and also break down for strong salt- or protein-induced deformations, as well as vesicle adhesion and fusion, which complicate the analysis of the lipid bilayer structure. Presented here are simulations of SAXS curves of SUVs with experimentally relevant size, shape and EDPs of the curved bilayer, inferred from coarse-grained simulations and elasticity considerations, to quantify the effects of size polydispersity, thermal fluctuations of the SUV shape and membrane asymmetry. It is observed that the factorization approximation of the scattering intensity holds even for small vesicle radii (∼30 nm). However, the simulations show that, for very small vesicles, a curvature-induced asymmetry arises in the EDP, with sizeable effects on the SAXS curve. It is also demonstrated that thermal fluctuations in shape and the size polydispersity have distinguishable signatures in the SAXS intensity. Polydispersity gives rise to low-q features, whereas thermal fluctuations predominantly affect the scattering at larger q, related to membrane bending rigidity. Finally, it is shown that simulation of fluctuating vesicle ensembles can be used for analysis of experimental SAXS curves.

Altering the intra-liver distribution of phospholipid-free small unilamellar vesicles using temperature-dependent size-tunability

We demonstrated that phospholipid-free small unilamellar vesicles (PFSUVs) composed of TWEEN 80 and cholesterol (25/75, mol%) could be fabricated using a staggered herringbone micromixer with precise controlling of their mean size between 54 nm and 147 nm. Increasing the temperature or decreasing the flow rate led to an increase in the resulting particle diameter. In zebrafish embryos, 120-nm PFSUVs showed 3-fold higher macrophage clearance compared to the 60-nm particles, which exhibited prolonged blood circulation. In mice, the 60-nm particles showed dominant accumulation in the liver hepatocytes (66% hepatocytes positive), while the 120-nm particles were delivered equally to the liver and spleen macrophages. Accordingly, in a murine model of acetaminophen-induced hepatotoxicity the 60-nm particles loaded with chlorpromazine reduced the serum alanine aminotransferase level and liver necrosis 2- to 4-fold more efficiently than their 120-nm counterparts and the free drug, respectively. This work showed that the intra-liver distribution of PFSUVs was largely determined by the size. Most other nanoparticles published to date are predominantly cleared by the liver Kupffer cells. The 60-nm PFSUVs, on the other hand, focused the delivery to the hepatocytes with significant advantages for the therapy of liver diseases.

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Membrane-dependent amyloid aggregation of human BAX α9 (173-192).

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

The docking of synaptic vesicles on the presynaptic membrane induced by \u03b1-synuclein is modulated by lipid composition

α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson’s disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.

Morphological and Nanomechanical Properties of Model of Animal and Human Eye Lens Lipid Membranes

The four major phospholipids found in eye lens membrane are phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Lens lipid composition of the fiber-cell membrane significantly changes with species. In lower-age animals, phosphatidylcholine (PC) is dominant, whereas, in humans, sphingomyelin (SM) is more dominant. We have used atomic force microscopy (AFM) to study morphological and nanomechanical properties of the lens lipid membrane models from mouse, pig, and humans. Small unilamellar vesicles (SUVs) were prepared from the mixture of four phospholipids (PC, SM, PS, and PE) that resemble mouse, pig, and human phospholipid composition lens membranes. The SUVs phospholipids mixtures were prepared with 0 mol% and 50 mol% of cholesterol. The topographical image and the mechanical properties of animal and human lens model membranes were accessed in a supported lipid membrane (SLM) formed on mica. The topographical images of phospholipid mixtures with 0 mol% cholesterol for both animal and human lens membrane models exhibited coexisting solid-ordered and liquid-disordered phase, where the proportion of solid-ordered phase increases with an increase in SM content in phospholipid mixture. The height difference between the two phases was ∼ 1nm. However, in the presence of 50 mol% of cholesterol, the membrane exhibited only a liquid-ordered phase, both for animal and human phospholipid mixtures. The breakthrough force on the SLM of the animals and human lens model membrane obtained with 50 mol% of cholesterol follow the trends: F (human) > F (pig) > F (mouse). Similarly, the membrane area compressibility modulus (Ka) of the membrane with 50 mol% cholesterol was accessed using the force curve's elastic regime. We found: Ka (human) > Ka (pig) > Ka (mouse). Our results show that phospholipid composition plays a significant role in modulating membrane mechanical properties.

Membrane Insertion of Intrinsically Disordered Proteins Probed by Rotational and Triplet State Dynamics

Small aggregates of intrinsically disordered proteins are thought to induce cytotoxicity by attaching to lipid membranes and by making them ion-permeable. Understanding the nature of such membrane bound oligomers has remained a challenge. Here we demonstrate a method to investigate the location of the different segments of the oligomers inside the lipid bilayer of small unilamellar lipid vesicles. We achieve this by measuring distance dependent modulation of the triplet state dynamics of fluorescence probes (placed on the peptides) by lipids labelled with radicals at different sites. Additionally, we measure the rotational dynamics of the fluorescent probes by measuring their long-timescale anisotropy using polarized fluorescence correlation spectroscopy (FCS). We observe that the triplet relaxation rate of a fluorophore placed on the N-terminal of amylin (hIAPP, associated with Type II diabetes) changes doubles in lipid vesicles which have a radical label on the C16 position of the alkyl chain, and changes less for other radical positions. We infer that the N-terminal is heterogeneously distributed in the membrane, though a major fraction inserts deep inside the lipid bilayer. The measurement of the rotational correlation times with polarized FCS shows strong similarity in mobility between both the N and the C termini. In contrast, Amyloid-beta has its N-terminal close to the C5 to C10 region of the lipid bilayer where the triplet relaxation rate becomes 1.4 and 1.5 times, respectively. We are currently studying the local mobility of different parts of these proteins in lipid membrane with electron paramagnetic resonance measurements and with molecular dynamics simulations. Our study will enhance our understanding of how these small aggregates are able to construct ion permeable channels in the membrane.

Interface interaction between high-siliceous/calcareous mineral granules and model cell membranes dominated by electrostatic force.

High-siliceous/calcareous mineral granules may cause cytotoxicity by attaching to cell membranes. In this research, giant (GUVs) and small unilamellar vesicles (SUVs) were used as model membranes for studying the interaction between high-siliceous/calcareous mineral granules (micro calcite, micro quartz, nano calcium carbonate, and nano silica) and artificial membranes. Confocal laser scanning microscopy (CLSM) and fluorescence labeling experiments suggest that nano calcium carbonate (nano CaCO3) and nano silica (nano SiO2) induce gelation by disrupting the oppositely charged membranes, indicating the important role of electrostatic forces. Thereby, the mineral granule size affects the electrostatic interactions and thus leading to the damage of the membranes. FTIR spectra and molecular dynamics reveal that mineral granules mainly interact with -PO2-, -OH, and -C-N(CH3)3+ groups in phospholipids. The electrostatic force between nano minerals and phospholipids is greater in the case SiO2 when compared to CaCO3. Moreover, nano SiO2 forms the strongest hydrogen bond with the -PO2- group as confirmed by FTIR. Thus, nano SiO2 causes the greatest damage to membranes. This research provides a deeper understanding of the mechanism regarding the interaction between inhalable mineral granules and cell membranes.

Supramolecular ultra-short carboxybenzyl-protected dehydropeptide-based hydrogels for drug delivery

Self-assembled peptide-based hydrogels are promising materials for biomedical research owing to biocompatibility and similarity to the extracellular matrix, amenable synthesis and functionalization and structural tailoring of the rheological properties. Wider developments of self-assembled peptide-based hydrogels in biomedical research and clinical translation are hampered by limited commercial availability allied to prohibitive costs.In this work a focused library of Cbz-protected dehydrodipeptides Cbz-L-Xaa-Z-ΔPhe-OH (Xaa= Met, Phe, Tyr, Ala, Gly) was synthesised and evaluated as minimalist hydrogels. The Cbz-L-Met-Z-ΔPhe-OH and Cbz-L-Phe-Z-ΔPhe-OH hydrogelators were comprehensively evaluated regarding molecular aggregation and self-assembly, gelation, biocompatibility and as drug carriers for delivery of the natural compound curcumin and the clinically important antitumor drug doxorubicin. Drug release profiles and FRET studies of drug transport into small unilamellar vesicles (as biomembrane models) demonstrated that the Cbz-protected dehydropeptide hydrogels are effective nanocarriers for drug delivery. The expedite and scalable synthesis (in 3 steps), using commercially available reagents and amenable reaction conditions, makes Cbz-protected dehydrodipeptide hydrogels, widely available at affordable cost to the research community.

Vesicle deformation and division induced by flip-flops of lipid molecules.

We investigated the deformation of small unilamellar vesicles (SUVs) induced by flip-flops of lipids using coarse-grained molecular dynamics simulations. In the case of single-component SUVs composed of zero spontaneous curvature lipids (ZLs), the flip-flop of ZLs deformed stomatocyte-shaped SUVs into an oblate shape, whereas pear-shaped SUVs were deformed into a prolate shape. These two equilibrium shapes comply with the local minima of elastic energy. In the case of binary vesicles composed of ZLs and negative spontaneous curvature lipids (NLs), the vesicle deformation pathway depended on the initial NL distribution in the bilayer. If the initial difference in the NL concentration between the outer and inner leaflets was small, the flip-flop of ZLs and NLs rapidly deformed pear-shaped SUVs into an equilibrium prolate shape. On the other hand, when NLs were localised in the inner leaflet, the flip-flop of ZLs and NLs deformed pear-shaped SUVs into a limiting shape and then induced vesicle division. Because the flip-flop rate of NLs is much faster than that of ZLs, the total free energy was first relaxed by the flip-flop of NLs and then by that of ZLs. This kinetic effect is responsible for the observed vesicle division induced by flip-flops.

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles.

The introduction of chirality into aqueous self-assemblies by employing isotactic block copolymers (BCPs) is an emerging field of interest as it promises special membrane properties of polymersomes not accessible by atactic BCPs. However, isotactic BCPs typically exhibit crystalline behaviour, inducing high membrane stiffness and limiting their applicability in systems involving membrane proteins or sensitive cargo. In this study, an isotactic yet fully amorphous BCP is introduced which overcomes these limitations. Three BCPs composed of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG), differing solely in their tacticities (R/S, R and S), were synthesised and characterised regarding their structural, optical and thermal properties. Their self-assembly into homogenous phases of nanoscopic polymersomes (referred to as small unilamellar vesicles, SUVs) was analysed, revealing stability differences between SUVs composed of the different BCPs. Additionally, microscopic giant unilamellar vesicles (GUVs) were prepared by double emulsion microfluidics. Only the atactic BCP formed GUVs which were stable over several hours, whereas GUVs composed of isotactic BCPs ruptured within several minutes after formation. The ability of atactic PBO-b-PG to form microreactors was elucidated by reconstituting the membrane protein OmpF in the GUV membrane by microfluidics and performing an enzyme reaction inside its lumen. The system presented here serves as platform to design versatile vesicles with flexible membranes composed of atactic or isotactic BCPs. Hence, they allow for the introduction of chirality into nano- or microreactors which is a yet unstudied field and could enable special biotechonological applications.

Flow Linear Dichroism of Protein-Membrane Systems.

Linear dichroism (LD) is the differential absorbance of light polarized parallel and perpendicular to an orientation direction. Any oriented sample will show a signal in its electronic as well as vibrational transitions. Model membrane small unilamellar vesicles or liposomes provide an oriented system when they are subject to shear flow in a Couette or other type of flow cell. Anything, including peptides and proteins, that is bound to the liposome also gives an LD signal whereas unbound analytes are invisible. Flow LD is the ideal technique for determining the orientation of different chromophores with respect to the membrane normal. To illustrate the power of the method, data for diphenyl hexatriene, fluorene, antimicrobial peptides (aurein 2.5 and gramicidin), are considered as well as another common chromophore, fluorene, often used to increase the hydrophobicity and hence membrane binding of peptides. How LD can be used both for geometry, structure analysis and probing kinetic processes is considered. Kinetic analysis usually involves identifying binding (appearance of an LD signal), insertion (sign change), often followed by loss of signal, if the inserted protein or peptide disrupts the membrane .

Discrete fluid lipid membrane compositional array created with chrome and protein barriers

To understand various interactions with cell membranes for in-vivo studies array of different lipid compositions becomes important. Work presented here shows how to prepare different lipid compositions for two different lipids with the help of micro-contact printing and photolithography. Firstly, barriers were created on hydrophilic surface by chrome patterning and/or by protein patterning. Secondly, lipid membrane of desired fluid lipid composition containing one or more lipids in it can be printed by micro-contact printing on to the surface containing chrome or protein patterns. It results in formation of 5 nm thick lipid membrane pattern partitioned with protein or chrome patterns. Thirdly, secondary intercalation can be done by adding small unilamellar vesicles of fluid lipid composition containing one or more lipids to the earlier patterned surface. Upon backfilling with the secondary vesicles it results in formation of compositional array lipid membrane patterns upon mixing with the existing patterned bilayer due to lateral fluidity.

Design, assembly, and characterization of membrane-spanning DNA nanopores.

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores' chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore-membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2-3 weeks for DNA origami pores.

LIPOSOMES – A REVIEW

The function of belayed vesicles as productive transporters for drugs, immunizations, indicative specialists, and other bioactive operators has prompted a fast headway in the liposomal drug conveyance system. The pharmacy- elements and pharmacokinetics properties are altered for the liposomal delivery system,which on thewholeleads to an increased the rapeutic index with decreased toxicity. The liposome can be named multilamellar vesicles or unilamellar vesicles, which can be additionally named large unilamellar vesicles (LUV) or small unilamellar vesicles (SUV). The part of liposome as a medication conveyance framework is to convey drug in a controlled way, diminishing unfortunate reactions improving it is in vitro and in vivo action, just as lessening the harmfulness of the medication and upgrading the viability of the exemplified drug. This article gives a review of techniques to the planning of liposome, just as diagnostic strategies for control physical, concoction, and organic boundaries for various kinds of medications.

Nanoliposomal Cercodemasoide A and Its Improved Activities Against NTERA-2 Cancer Stem Cells

Recently, saponins derived from marine sources have received much attention because of their promising bioactivities, such as anticancer, anti-angiogenesis, and anti-inflammation. In particular, a triterpene saponin from the sea cucumber Cercodemas anceps Selenka, cercodemasoide A (CAN1), showed potent cytotoxicity against various cancer cell lines. Recent evidence has indicated that cancer stem cells (CSCs) could be a novel target for efficient cancer therapies. In order to improve the biopharmaceutical properties of CAN1, the compound was loaded into nanoliposomes as an ideal drug carrier. CAN1 was successfully incorporated into nanoliposomes as small unilamellar liposome vesicles with an average size of 73.39 ± 1.57 nm, zeta potential of −0.299 ± 0.046 mV, polydispersity index of 0.336 ± 0.038, and with an encapsulation efficiency of up to 62.9%. For the first time, CAN1 and its nanoliposomal forms have been shown to have a promising cytotoxic activity against NTERA-2 CSCs, with half-maximal inhibitory concentration (IC50) =1.03 ± 0.04 and 0.41 ± 0.03 µM, respectively. The CAN1 nanoliposomes also presented significantly improved activities in suppressing the growth of NTERA-2 3-dimensional tumorspheres (IC50 = 1.71 ± 0.06 µM) in comparison with the free form ( P < .05). The anti-CSC effects of CAN1 nanoliposomes on NTERA-2 cells were due to their apoptotic induction through enhancing caspase-3 activity (more than 2-fold) and arresting the cell cycle at the S phase ( P < .05). The obtained CAN1-encapsulated nanoliposomes suggest valuable applications in CSC-targeting treatment for more efficient clinical therapy.

Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.

Theranostic verteporfin- loaded lipid-polymer liposome for photodynamic applications

In this study we report a novel theranostic lipid-polymer liposome, obtained from DPPC and the triblock copolymer F127 covalently modified with 5(6)-carboxyfluorescein (CF) for photodynamic applications. Due to the presence of F127, small unilamellar vesicle (SUV) liposomes were synthesized by a simple and fast thin-film hydration method without the need for an extrusion process. The vesicles have around 100 nm, low polydispersity and superb solution stability. The clinically used photosensitizer verteporfin (VP) was entrapped into the vesicles, mostly in monomeric form, with 90% loading efficiency. Stern-Volmer and fluorescence lifetime assays showed heterogeneous distribution of the VP and CF into the vesicles, ensuring the integrity of their individual photophysical properties. The theranostic properties were entirely photoactivatable and can be trigged by a unique wavelength (470 nm). The feasibility of the system was tested against the Glioblastoma multiforme cell line T98G. Cellular uptake by time-resolved fluorescence microscopy showed monomerized VP (monoexponential decay, 6.0 ns) at nucleus level, while CF was detected at the membrane by fluorescence microscopy. The strategy's success was supported by the reduction of 98% in the viability of T98G cells by the photoactivated lipid-polymer liposome with [VP] = 1.0 μmol L−1. Therefore, the novel theranostic liposome is a potential system for use in cancer and ocular disease therapies.