Calcein Leakage Research Articles

Pore-forming activity of new conjugate antibiotics based on amphotericin B.

A series of amides of the antifungal antibiotic amphotericin B (AmB) and its conjugates with benzoxaboroles was tested to determine whether they form pores in lipid bilayers and to compare their channel characteristics. The tested derivatives produced pores of larger amplitude and shorter lifetime than those of the parent antibiotic. The pore conductance was related to changes in the partial charge of the hydrogens of the hydroxyl groups in the lactone ring that determined the anion coordination in the channel. Neutralization of one of the polar group charges in the AmB head during chemical modification produced a pronounced effect by diminishing the dwell time of the polyene channel compared to modification of both groups. In this study, compounds that had a modification of one carboxyl or amino group were less effective in initializing phase separation in POPC-membranes compared to derivatives that had modifications of both polar groups as well as the parent antibiotic. The effects were attributed to the restriction of the aggregation process by electrical repulsion between charged derivatives in contrast to neutral compounds. The significant correlation between the ability of derivatives to increase the permeability of model membranes—causing the appearance of single channels in lipid bilayers or inducing calcein leakage from unilamellar vesicles—and the minimal inhibitory concentration indicated that the antifungal effect of the conjugates was due to pore formation in the membranes of target cells.

Calcein Release from Cells In Vitro via Reversible and Irreversible Electroporation.

The aim of this study was to investigate the dependence of calcein extraction and cell viability on the parameters of pulsed electric field (PEF). Two different approaches concerning PEF parameters were investigated: (1) extraction efficiency and cell viability dependence on pulse number, exploiting 1200V/cm 100 µs duration high voltage (HV) electric pulses and (2) extraction efficiency and cell viability dependence on the pulses with different duration (44-400 µs) and electric field strength (600-1800V/cm) that result in the same amount of electric field energy delivered to Chinese hamster ovary cells. Extraction efficiency was evaluated as a percentage ratio of calcein fluorescence intensity prior and after PEF treatment. Cell viability was evaluated using PI test and cell clonogenic assay. Moreover, calcein release dynamics from cells after 600V/cm 400 µs, 1200V/cm 100 µs, and 1800V/cm 44 µs was evaluated. Our results show that HV pulses induce instant calcein extraction due to reversible electroporation; however, subsequent calcein leakage over time was only observed when 9 HV pulses of 1800V/cm 44 µs were used. The increased number of pulses resulted in more efficient total calcein extraction. With the same total energy delivered via electric pulses, the increase of calcein extraction efficiency was more dependent on pulse strength rather than pulse duration. The highest calcein extraction efficiency (84.5 ± 7.4%) was obtained using 9 electric field pulses of 1800V/cm, 44 µs at 1Hz. Furthermore, the extraction efficiency can be significantly enhanced if external mechanical stress (pipetting) is applied to cells. Cell viability was determined to be dependent on different PEF exposure parameters. It varied from 96.8 ± 4.8 to 31.2 ± 8.9%, implying the possibility to adjust PEF parameter combinations to maintain high cell viability.

A Cell-Mimicking Structure Converting Analog Volume Changes to Digital Colorimetric Output with Molecular Selectivity.

We herein report a three-component cell-mimicking structure with a peroxidase-like iron oxide nanozyme as the nucleus, a molecularly imprinted hydrogel shell as cytoplasm, and a lipid bilayer membrane. The structure was characterized by cryo and negative stain TEM and also by a calcein leakage test. By introducing charged monomers, the gel shell can swell or shrink in response to salt concentration. By lowering the salt concentration, the gradual "analog" gel volume change was reflected in a switch-like "digital" colorimetric output by the burst of membrane and oxidation of substrates such as 3,3',5,5'-tetramethylbenzidine (TMB). Controlled access was also achieved by using melittin to insert channels cross the membrane, and selective molecular transport was realized by the molecularly imprinted gel. The functions of each component are coupled, and this sophisticated tripartite structure provides a new platform for modular design of new materials. Our cell-mimicking structure is functional and it is complementary to the current protocell work that aims to understand the origin of life.

Differential Effect of Bilayer Thickness on Sticholysin Activity.

In this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using dimonounsaturated phosphatidylcholine (PC) analogues (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUVs) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn-LUV interaction, we observed that the bilayer affinity constant (Ka) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins' N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC.

Isothermal titration calorimetry and vesicle leakage assays highlight the differential behaviors of tau repeat segments upon interaction with anionic lipid membranes

Tau misfolding has been implicated in a variety of tauopathies, including Alzheimer's disease. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments leads to the formation of neurofibrillary tangles. Previous studies indicate that misfolded tau associates with anionic phospholipid membranes, invoking structural transformations that could play a role in aggregation. Here, we investigated the role of membrane surface charge on the binding affinity of individual tau repeat segments, and whether these segments exhibit lytic activity. We quantified the thermodynamics of this process in terms of the affinity (Kd), enthalpy (ΔH), entropy (ΔS), and change in specific heat capacity (ΔCp). While neutral membranes exhibited weak interactions with each tau repeat segment, segments R2 and R3 exhibited relatively strong binding with anionic membranes with favorable ΔS and a negative value of ΔCp. Calcein leakage assays show that each repeat segment displays lytic activity, but only upon the interaction with anionic membranes. Taken together, these results distinguish the relative selectivity for anionic membranes by each repeat segment and the degree of membrane disruption that results.

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut

BackgroundThe importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian β-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds.ResultsTime-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05).ConclusionAmino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Freeze-dryable lipid vesicles with size tunability and high encapsulation efficiency prepared by the multiple emulsification-solvent evaporation method

We investigated the extent of potential applicability of our recently developed method for preparing lipid vesicles [T. Kuroiwa et al., J. Am. Oil Chem. Soc., 93 (2016) 421], designated as the multiple emulsification-solvent evaporation method, with the intention of controlling the vesicle diameter and achieving high entrapment efficiency for water-soluble compounds. Using this method, the diameter of lipid vesicles could be varied by selecting the methods for preparing the primary water-in-oil emulsion, which contained water droplets as templates for the internal water phases of lipid vesicles. We obtained lipid vesicles with mean diameters of 0.2–4.4μm from water-in-oil-in-water multiple emulsions after solvent evaporation. A high entrapment yield of calcein, a water-soluble fluorescent dye, into the lipid vesicles was obtained for each vesicle sample, depending on their diameter and the type of emulsifier added to the external water phase. The use of polymeric emulsifier was more effective in achieving a high entrapment yield. The obtained lipid vesicles were powderized via freeze-drying. Vesicles could be powderized while maintaining their original diameter, as confirmed by scanning electron microscopy. Furthermore, the powderized vesicles could be rehydrated and resuspended without significant change in their diameter. However, the entrapment yield of calcein decreased after freeze-drying and rehydration. The calcein leakage during the freeze-drying followed by rehydration could be suppressed by adding an appropriate amount of trehalose as a lyoprotectant.

Calcein Leakage Behavior from Vesicles Induced by Protein–Vesicle Interaction: A Study by Surface Pressure–Area Isotherms

The release behavior of substrates encapsulated in the vesicle can be controlled by the interaction between the vesicular membrane and the protein. However, its control mechanism is still now unknown. Therefore, the protein–vesicle interaction was investigated by a π–A isotherm. It was clarified that the released amount of encapsulated substrates increased with increasing protein–vesicle interaction. This result leads to the development of drug delivery systems (DDS) and of biosensor technology.

Calcein leakage as a robust assay for cytochrome c/H2O2–mediated liposome permeabilization

Membrane-permeabilizing activity of cytochrome c (cyt c) in the presence of hydrogen peroxide associated with its functioning as peroxidase is considered relevant to initiation of the mitochondrial pathway of apoptosis. Here, we present evidence that the choice of a fluorescent dye for measuring cyt c/H2O2-induced dye leakage from liposomes by fluorescence de-quenching is of major importance. The popular fluorescent marker 5(6)-carboxyfluorescein appeared highly susceptible to cyt c-mediated peroxidative destruction and therefore unsuitable for the leakage assay with cyt c/H2O2. On the contrary, calcein, another conventional marker, proved resistant to oxidative stress and thus perfectly suitable for the assay. Based on the concentration dependences of the cyt c/H2O2-induced calcein leakage, the optimal conditions for the assay were found.

Synthetic toxic Aβ1–42 oligomers can assemble in different morphologies

BackgroundAlzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aβ peptides. Aβ toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aβ1–42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. MethodsDifferent biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used. ResultsThe oligomers are mostly populated by a 14mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aβ amyloid fibrils. ConclusionThe toxic synthetic AβG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel β-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aβ dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes. General significanceThe capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.

Electrostatic Interactions between Tau Peptides and Model Biological Membranes: A Folding-Upon-Binding Mechanism

Found in neuronal cells of the central nervous system, tau proteins primarily function in cytoskeletal stability by associating with microtubules. During Alzheimer's disease and other tauopathies, misfolded tau proteins form neurofibrillary tangles (NFTs), thus impairing neuronal function and causing various neurodegenerative effects. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments has previously been observed within NFTs. Previous research has also found misfolded tau components associated with the Golgi membrane, but not the plasma membrane. Here, we have used circular dicroism (CD) and isothermal titration calorimetry (ITC) to examine interactions between repeat segments R1-R4 and lipid bilayers of varying charge (DOPC and DOPC/DOPG 80:20). Our data indicate that significant structural changes are induced by charged membrane mimics and an entropy-driven binding process occurs during this interaction. Indeed, binding of tau occurs concomitantly with a classical hydrophobic effect. Thus, we propose a folding-upon-binding event occurring between negatively-charged membranes and the more promiscuous tau repeat segments. Results from calcein leakage assays and membrane fusion FRET measurements will also be presented to further probe the mechanism of bilayer disruption. These results will help explain a possible mechanism of tau misfolding during Alzheimer's disease, and are potentially beneficial to design strategies that prevent or reverse the aggregation process.

Adsorption of Nanoceria by Phosphocholine Liposomes.

Nanoceria (CeO2 nanoparticle) possesses a number of enzyme-like activities. In particular, it scavenges reactive oxygen species based on in vitro and in vivo antioxidation studies. An important aspect of fundamental physical understanding is its interaction with lipid membranes that are the main components of the cell membrane. In this work, adsorption of nanoceria onto phosphocholine (PC) liposomes was performed. PC lipids are the main constituents of the cell outer membrane. Using a fluorescence quenching assay, a nanoceria adsorption isotherm was determined at various pH values and ionic strengths. A non-Langmuir isotherm occurred at pH 4 because of lateral electrostatic repulsion among the adsorbed cationic nanoceria. The phosphate group in the PC lipid is mainly responsible for the interaction, and the adsorbed nanoceria can be displaced by free inorganic phosphate. The tendency of the system to form large aggregates is a function of pH and the concentration of nanoceria, attributable to nanoceria being positively charged at pH 4 and neutral at physiological pH. Calcein leakage tests indicate that nanoceria induces liposome leakage because of transient lipid phase transition, and cryo-transmission electron microscopy indicates that the overall shape of the liposome is retained although deformation is still observed. This study provides fundamental biointerfacial information at a molecular level regarding the interaction of nanoceria and model cell membranes.

The Solvent-Exposed C-Terminus of the Cytolysin A Pore-Forming Toxin Directs Pore Formation and Channel Function in Membranes.

Pore-forming toxins (PFTs) bind to cell membranes and form nanoscale pores that allow leakage of cellular components, resulting in cell death. The water-soluble, monomeric form of these toxins shows a dramatic conformational change during pore formation, as exemplified by crystal structures of the monomer and functional pore of cytolysin A (ClyA). The solvent-exposed, C-terminal residues of the protein are essential for activity, but the mechanism by which this region regulates pore formation remains unknown. We show here that deletion of the C-terminus of ClyA did not alter its ability to bind to the membrane or oligomerize in detergent. However, the truncated toxin lysed erythrocytes poorly, was more susceptible to proteolysis and thermal unfolding, and showed low calcein leakage from small unilamellar vesicles. Using fully atomistic molecular dynamics (MD) simulations, we find that deletion of C-terminal residues from the ClyA monomer significantly altered stability and unfolding trajectories in the transmembrane N-terminal helix, a region that is pivotal in maintaining the structural integrity of the helical bundle. MD simulations of pores with or without the C-terminus showed minor differences, implying that if oligomerization could be induced prior to the addition to vesicles, then an active pore could be generated. Via generation of oligomers in a detergent prior to the addition to vesicles, the truncated toxin could induce calcein leakage from vesicles, albeit to a lower extent. Therefore, regions of pore-forming toxins, not directly involved in the pore structure, are not passive players but have important roles in undergoing the transition through intermediary steps leading to successful pore formation in a membrane environment.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins.

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo-TEM, and ζ-potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3 O4 , TiO2 , ZrO2 , Y2 O3 , ITO, In2 O3 , and Mn2 O3 ) form supported bilayers only with DOCP. Type 2 (SiO2 ) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3 O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins

AbstractMetal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo‐TEM, and ζ‐potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3O4, TiO2, ZrO2, Y2O3, ITO, In2O3, and Mn2O3) form supported bilayers only with DOCP. Type 2 (SiO2) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.

Activity of tick antimicrobial peptide from Ixodes persulcatus (persulcatusin) against cell membranes of drug-resistant Staphylococcus aureus

Persulcatusin (IP), which is an antimicrobial peptide found in Ixodes persulcatus midgut, is active against Gram-positive bacteria such as Staphylococcus aureus. Multidrug-resistant bacteria in particular methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are a worldwide clinical concern. In the present study, to explore the potential of IP as a new agent against multidrug-resistant S. aureus infections, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains by MIC90, morphological observation with scanning electron microscope (SEM), and the calcein leakage assay of membrane integrity. Among the six antimicrobial peptides used in this study, IP exhibited the lowest MIC90 values for both vancomycin-susceptible and -resistant S. aureus strains. The IP MIC90 against a VISA strain was equivalent to vancomycin, while the MIC90 against VRSA was relatively low. SEM observations indicated that bacterial cells exposed to IP were crumpled and showed prominent structural changes. Moreover, IP influenced the cell membranes of both MRSA and VRSA in a mere 5 min, leading to leakage of the preloaded calcein. Although a VISA strain was resistant to the action of IP on cell membrane, the MIC90 of IP was lower than that of Nisin, suggesting that IP had another bactericidal mechanism in addition to cell membrane attack. Our results indicate that the synthetic tick antimicrobial peptide, IP exhibits strong antibacterial activity against multidrug-resistant S. aureus strains, including VRSA, via both cell membrane attack and another unknown mechanism. IP represents a promising candidate for a new anti-VRSA therapy.

Affinity and selectivity of anchovy antibacterial peptide for Staphylococcus aureus cell membrane lipid and its application in whole milk

An anchovy antibacterial peptide ACWWP1 (GLSRLFTALK) has been previously shown to kill bacterial cells via membrane damage. The affinity and selectivity of ACWWP1 for cell membrane lipids and their effects on the activity were investigated. Thermodynamic analysis indicated that hydrophobic and electrostatic effects contributed to the partitioning of ACWWP1 in the membrane lipids. Confocal Raman microspectroscopy, fluorescence anisotropy and calcein leakage analysis suggested that the peptide induced the greatest change in the order parameters of the Staphylococcus aureus cell membrane, which was related to the relaxation of the lateral stacking and an increase in the fluidity of the phospholipid bilayer. The positively charged ACWWP1 could change the surface charge state of the membrane. Besides, ACWWP1 exhibited efficient antimicrobial activity for S. aureus in whole milk. The results suggest that ACWWP1 exhibits favorable bacterial cell membrane lipid selectivity and can be employed as an efficient method for the treatment of food poisoning caused by certain bacteria.

An increase in model lipid membrane fluidity as a result of local anesthetic action

The effect of local anesthetics on the permeability of phospholipid liposomes of different composition for calcein has been investigated. The local anesthetics tested included amides (lidocaine, prilocaine, mepivacaine, and bupivacaine) and esters (benzocaine, procaine, and tetracaine). The permeability of large monolamellar liposomes was assessed by monitoring the fluorescence of calcein leaking from the phospholipid vesicles. All tested amide anesthetics exerted negligible effects on the permeability of dioleylphosphocholine (DOPC) liposomes for the fluorescent marker. The most efficient in this group was did bupivacaine. Amides had a more pronounced effect on membranes in which 20 mol % of DOPC was replaced by tetraoleoylcardiolipin (TOCL). Benzocaine and procaine at concentration up to 100 mM did not affect the permeability of DOPC liposomes. Membrane permeability of DOPC liposomes was not affected by the addition of tetracaine to the final concentration of 2 mM, while the increase of anesthetic concentration up to 50 mM was accompanied by an increase in the intensity of fluorescence of calcein released from the vesicles, and addition of the anesthetic to the concentration of 100 mM caused by complete release of the marker incorporated by the liposomes. The threshold concentration of tetracaine initiating calcein leakage from vesicles that contained 20 mol % TOCL was 7 mM, and the concentration corresponding to 100% calcein leakage was 20 mM. Confocal fluorescence microscopy of giant monolamellar liposomes formed from an equimolar mixture of DOPC and tetramiristoylcardiolipin demonstrated the destruction of solid ordered domains at the presence of anesthetics, and its destructive capacity increasing in the following order: procaine ≈ mepivacaine < bupivacaine ≪ tetracaine. Variability of the depth of anesthetic incorporation into the membrane may account for the dissimilar effects of local anesthetics on liposomes.

Simple surface functionalization of polymersomes using non-antibacterial peptide anchors.

BackgroundHollow vesicles formed from block copolymers, so-called polymersomes, have been extensively studied in the last decade for their various applications in drug delivery, in diagnostics and as nanoreactors. The immobilization of proteins on the polymersomes’ surface can aid in cell targeting, lead to functional biosensors or add an additional reaction space for multistep syntheses. In almost all surface functionalization strategies to date, a chemical pre-conjugation of the polymer with a reactive group or ligand and the functionalization of the protein are required. To avoid chemical pre-conjugation, we investigated the simple and quick functionalization of preformed poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) polymersomes through the spontaneous insertion of four hydrophobic, non-antibacterial peptide anchors into the membrane to display enhanced green fluorescent protein (eGFP) on the polymersomes’ surface.ResultsThree of the four hydrophobic peptides, the transmembrane domains of a eukaryotic cytochrome b5, of the viral lysis protein L and of the yeast syntaxin VAM3 could be recombinantly expressed as soluble eGFP-fusion proteins and spontaneously inserted into the polymeric membrane. Characterization of the surface functionalization revealed that peptide insertion was linearly dependent on the protein concentration and possible at a broad temperature range of 4–42 °C. Up to 2320 ± 280 eGFP molecules were immobilized on a single polymersome, which is in agreement with the calculated maximum loading capacity. The peptide insertion was stable without disrupting membrane integrity as shown in calcein leakage experiments and the functionalized polymersomes remained stable for at least 6 weeks.ConclusionThe surface functionalization of polymersomes with hydrophilic proteins can be mediated by several peptide anchors in a spontaneous process at extremely mild insertion conditions and without the need of pre-conjugating polymers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0205-x) contains supplementary material, which is available to authorized users.