Permeability Of Liposomal Membranes Research Articles

Azobenzene-based liposomes with nanomechanical action for cytosolic chemotherapeutic drug delivery

The stimuli-responsive nano-carriers are at the forefront of research in nanotechnology and materials science. These advanced systems are designed to alter their physicochemical properties upon exposure to specific stimuli, enabling controllable and targeted delivery of therapeutic agents. Nevertheless, limited endosomal escape reduces the drug bioavailability in clinical use. We herein report azobenzene (Azo)-based liposomes, prepared by co-assembling the photoisomerizable cationic Azo lipids and helper lipids, which achieve controllable doxorubicin (Dox) release and enhanced cytosolic transport upon light irradiation. Azo lipids undergo reversible isomerization between cis-isomers and trans-isomer when received UV and visible (Vis) light irradiation, causing liposomal membrane permeability changes for controlled drug release. Moreover, the nanomechanical action created by the isomerization of Azo lipids promotes the endosomal escape of the liposomes. DSPC-Azo liposomes, with minimal Dox leakage, showed significant tumor cell killing upon irradiation. For in vivo study, we co-encapsulated the upconverting nanoparticles (UCNPs), which can convert the near-infrared (NIR) light into UV/Vis emissions, facilitating Azo units activation. UCNP/Dox-loaded DSPC-Azo liposomes inhibited tumor growth under NIR irradiation in a 4T1 tumor-bearing mouse model.

Spectroscopy Study of Albumin Interaction with Negatively Charged Liposome Membranes: Mutual Structural Effects of the Protein and the Bilayers.

Liposomes as drug carriers are usually injected into the systemic circulation where they are instantly exposed to plasma proteins. Liposome-protein interactions can affect both the stability of liposomes and the conformation of the associated protein leading to the altered biodistribution of the carrier. In this work, mutual effects of albumin and liposomal membrane in the course of the protein's adsorption were examined in terms of quantity of bound protein, its structure, liposome membrane permeability, and changes in physicochemical characteristics of the liposomes. Fluorescence spectroscopy methods and Fourier transform infrared spectroscopy (ATR-FTIR), which provides information about specific groups in lipids involved in interaction with the protein, were used to monitor adsorption of albumin with liposomes based on egg phosphatidylcholine with various additives of negatively charged lipidic components, such as phosphatidylinositol, ganglioside GM1, or the acidic lipopeptide. Less than a dozen of the protein molecules were tightly bound to a liposome independently of bilayer composition, yet they had a detectable impact on the bilayer. Albumin conformational changes during adsorption were partially related to bilayer microhydrophobicity. Ganglioside GM1 showed preferable features for evading undesirable structural changes.

Cyclodextrins and Their Polymers Affect the Lipid Membrane Permeability and Increase Levofloxacin's Antibacterial Activity In Vitro.

Cyclodextrins (CDs) are promising drug carriers that are used in medicine. We chose CDs with different substituents (polar/apolar, charged/neutral) to obtain polymers (CDpols) with different properties. CDpols are urethanes with average Mw of ~120 kDa; they form nanoparticles 100-150 nm in diameter with variable ζ-potential. We studied the interaction of CD and CDpols with model (liposomal) and bacterial membranes. Both types of CD carriers cause an increase in the liposomal membrane permeability, and for polymers, this effect was almost two times stronger. The formation of CD/CDpols complexes with levofloxacin (LV) enhances LV's antibacterial action 2-fold in vitro on five bacterial strains. The most pronounced effect was determined for LV-CD complexes. LV-CDs and LV-CDpols adsorb on bacteria, and cell morphology influences this process dramatically. According to TEM studies, the rough surface and proteinaceous fimbria of Gram-negative E. coli facilitate the adsorption of CD particles, whereas the smooth surface of Gram-positive bacteria impedes it. In comparison with LV-CDs, LV-CDpols are adsorbed 15% more effectively by E. coli, 2.3-fold better by lactobacilli and 5-fold better in the case of B. subtilis. CDs and CDpols are not toxic for bacterial cells, but may cause mild defects that, in addition to LV-CD carrier adsorption, improve LV's antibacterial properties.

(Invited) Detection of Bacterial Rhamnolipid Toxin By Redox Liposome Single Impact Electrochemistry

The single-entity impact method is a very sensitive and powerful electroanalytical tool for biological and environmental purposes such as electrochemical detection of pathogens.[1] The versatility of ultramicroelectrodes (UMEs) gives unique and essential properties for electroanalytical sensing owing to their small size, high sensitivity, fast steady-state response, low double-layer charging current and minimal ohmic loss. The observation of these stochastic events can provide information on various single nanoparticles contrary to ensemble (bulk) measurements. The main advantage of studying collisions of single entities is the low limit of detection (in principle, one single species) inherent to this electroanalytical method and the ability to study single entities (cells, viruses, nanoparticles...) in real time (dynamic measurement).[2–4]Electrochemistry of single redox liposome impacts at an UME consists in detecting the electrolysis of a redox probe encapsulated inside a liposome when it is released at the UME after impact (or collision). The UME is polarized at the oxidation or reduction potential of the encapsulated redox probe and a concentration of several pico-molar of redox liposomes added to the aqueous buffer electrolyte is enough to detect “current spikes” in the chronoamperometry (i-t) curve corresponding to discrete collision events.[3,4] The electron transfer does not readily occur across a lipid bilayer, thus the electrolysis of the liposome redox active content after collision and membrane rupture or opening at the UME surface led to many studies dealing with the membrane permeation mechanism.[3,4]Here, our work is based on the previous results where no current spike was observed in the chronoamperometry curve because the redox DMPC liposomes did not break during impact (or collision) onto the UME surface.[3,4] Hence, the electrochemical sensing principle is based on the weakening of the liposomes lipid membrane upon interaction with a destructive bacterial virulence factor which leads upon impact at an UME to the breakdown of the liposomes and the release/electrolysis of its encapsulated redox probe, as previously reported.[5] In the presence of RL toxin in solution (acting like a surfactant in the lipid membrane), current spikes corresponding to the electrolysis of the encapsulated redox probe released from weakened liposomes are detected (see Figure). Thanks to the redox liposome single impact electrochemistry, the highest detection limit of RL toxin (500 nM) has been reached in comparison to several micromoles per liter previously reported.[5][1] Lebègue, E.; Costa, N.L.; Louro, R.O.; Barrière, F. Communication—Electrochemical Single Nano-Impacts of Electroactive Shewanella Oneidensis Bacteria onto Carbon Ultramicroelectrode. J. Electrochem. Soc. 2020, 167, 105501, doi:10.1149/1945-7111/ab9e39.[2] Dick, J.E.; Lebègue, E.; Strawsine, L.M.; Bard, A.J. Millisecond Coulometry via Zeptoliter Droplet Collisions on an Ultramicroelectrode. Electroanalysis 2016, 28, 2320–2326, doi:10.1002/elan.201600182.[3] Lebègue, E.; Anderson, C.M.; Dick, J.E.; Webb, L.J.; Bard, A.J. Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode. Langmuir 2015, 31, 11734–11739, doi:10.1021/acs.langmuir.5b03123.[4] Lebègue, E.; Barrière, F.; Bard, A.J. Lipid Membrane Permeability of Synthetic Redox DMPC Liposomes Investigated by Single Electrochemical Collisions. Anal. Chem. 2020, 92, 2401–2408, doi:10.1021/acs.analchem.9b02809.[5] Luy, J.; Ameline, D.; Thobie-Gautier, C.; Boujtita, M.; Lebègue, E. Detection of Bacterial Rhamnolipid Toxin by Redox Liposome Single Impact Electrochemistry. Angew. Chem. Int. Ed. 2021, accepted , doi: 10.1002/anie.202111416. Figure 1

Single Electrochemical Nano-Impacts of Synthetic Redox Phospholipid Liposomes

Electrochemical discrete nano-impacts at an ultramicroelectrode is a useful technique to detect one at a time single biological entities such as cells, bacteria, macromolecules, viruses, and synthetic or biological vesicles.[1-4] Especially, electrochemical detection of single liposome nano-impacts by recording electron transfer from the ultramicroelectrode to an encapsulated redox species is fully appropriate for studying their membrane permeability.[2,4] Since electron transfer do not readily occur across a bilayer lipid membrane, the electrolysis of the liposome redox active content after collision and membrane rupture or opening at the electrode surface provides insights on the membrane permeation mechanism. The vesicle membrane opening by electroporation is strongly dependent on lipid membrane properties, liposome content, vesicle size, temperature, electrode potential, the nature of the electrode and the concentration of redox species inside and outside the liposome.[2] For example, to observe the current spikes corresponding to the oxidation of ferrocyanide encapsulated inside phospholipid vesicles when they collide with a Pt ultramicroelectrode, the presence of an appropriate concentration of surfactant in solution is required.[4] In the absence of surfactant, we found that impact and adhesion of vesicles at the Pt ultramicroelectrode does not allow the electrolysis of their ferrocyanide content.[4] It is well established that the liposome membrane stability is strongly dependent on its lipid composition and external parameters such as temperature and pH but also depends on interactions with specific molecules able to weaken, permeabilize, and penetrate the lipid bilayer following different pathways. An understanding of these different interactions and especially the mechanism leading to the lipid membrane opening is still an active research field. To this end, chronoamperometry is a useful method to probe the liposomes membrane permeability and to understand vesicle fusion processes onto electrode surface. Especially, the electrochemical detection of single liposome collisions at ultramicroelectrodes is becoming an efficient and complementary tool for analyzing fundamental biological processes related to intracellular and extracellular electron transfers to discrete biological or artificial entities. Electrochemistry of single redox liposome nano-impacts at an ultramicroelectrode is a powerful method which consists in detecting the electrolysis of a redox probe encapsulated inside the liposome when it is released at the ultramicroelectrode after impact (or collision).[4]To explore the factors influencing the vesicles membrane permeability, we investigated the electrochemical and electrocatalytic reaction of different aqueous redox species (potassium ferrocyanide and cobalt(II) nitrate) encapsulated inside synthetic DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) liposomes subjected to different experimental conditions (addition of surfactant, increase of temperature or presence of a redox probe in solution), by single collision detection on 10 μm diameter ultramicroelectrodes (Pt and carbon) as illustrated in Figure.[2] The influence of the presence of surfactant (Triton X-100) and the increase of solution temperature (20-70 °C) on the lipid membrane permeability has been investigated, and the results showed a similar effect of these two parameters with a maximum collision frequency reached for an optimal surfactant concentration (0.2 mM) and temperature (60 °C) respectively, at platinum or carbon ultramicroelectrodes.[2][1] Lebègue, E.; Costa, N.L.; Louro, R.O.; Barrière, F. Communication—Electrochemical Single Nano-Impacts of Electroactive Shewanella Oneidensis Bacteria onto Carbon Ultramicroelectrode. J. Electrochem. Soc. 2020, 167, 105501, doi:10.1149/1945-7111/ab9e39.[2] Lebègue, E.; Barrière, F.; Bard, A.J. Lipid Membrane Permeability of Synthetic Redox DMPC Liposomes Investigated by Single Electrochemical Collisions. Anal. Chem. 2020, 92, 2401–2408, doi:10.1021/acs.analchem.9b02809.[3] Dick, J.E.; Lebègue, E.; Strawsine, L.M.; Bard, A.J. Millisecond Coulometry via Zeptoliter Droplet Collisions on an Ultramicroelectrode. Electroanalysis 2016, 28, 2320–2326, doi:10.1002/elan.201600182.[4] Lebègue, E.; Anderson, C.M.; Dick, J.E.; Webb, L.J.; Bard, A.J. Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode. Langmuir 2015, 31, 11734–11739, doi:10.1021/acs.langmuir.5b03123. Figure 1

Effect of Methyltriclosan on the Functioning of Isolated Rat Liver Mitochondria and Permeability of Liposomal Membranes

Effect of Methyltriclosan on the Functioning of Isolated Rat Liver Mitochondria and Permeability of Liposomal Membranes

Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy

Naturally sourced phospholipids are used in many liposomal pharmaceuticals. The present report describes a method to detect the effects of different egg yolk phosphatidylcholines (EPCs) on liposomal physicochemical properties. Five EPC-containing liposomes were prepared using five different EPCs obtained from different suppliers. There was no significant difference in purity between each EPC. The stiffness of the liposomes was examined via atomic force microscopy (AFM) in relation to the liposomal membrane permeability coefficient of encapsulated calcein after gel filtration, which is indicative of liposomal stability including the release of a hydrophilic drug from a liposome. Although the size of the liposome and the encapsulation efficiency of calcein did not significantly change with the type of EPC used, the liposome stiffness was found to vary depending on the EPC used, and liposomes with a similar stiffness were found to show a similar membrane permeability to calcein. Our results indicate the usefulness of stiffness measurement, using AFM as the analytical method, to detect material-derived differences in EPC-containing liposomes that affect drug release from the liposomes. Because drug release is one of the most important liposomal functions, combining this method with other analytical methods could be useful in selecting material for the development and quality control of EPC-containing liposomes.

Liposomal membrane permeability assessment by fluorescence techniques: Main permeabilizing agents, applications and challenges.

Liposomes are lipid vesicles made of one or multiple lipid bilayers surrounding an internal aqueous core. They are broadly employed as models to study membrane structure and properties. Among these properties, liposome membrane permeability is crucial and widely assessed by fluorescence techniques. The first part of this review is devoted to describe the various techniques used for membrane permeability assessment. Attention is paid to fluorescence techniques based on vesicle leakage of self-quenching probes, dye/quencher pair or cation/ligand pair. Secondly, the membrane-active agents inducing membrane permeabilization is presented and details on their mechanisms of action are given. Emphasis is also laid on the intrinsic and extrinsic factors that can modulate the membrane permeability. Hence, a suitable liposomal membrane should be formulated according to the aim of the study and its application.

Lipid Membrane Permeability of Synthetic Redox DMPC Liposomes Investigated by Single Electrochemical Collisions.

The electrochemical detection of synthetic redox DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) liposomes by single collisions at 10 μm diameter carbon and Pt ultramicroelectrodes (UMEs) is reported. To study the parameters influencing the lipid membrane opening/permeability, the electrochemical detection of single redox DMPC liposome collisions at polarized UMEs was investigated under different experimental conditions (addition of surfactant, temperature). The electrochemical responses recorded showed that the permeability of the DMPC lipid membrane (tuned by addition of Triton X-100 surfactant or by the increase of the solution temperature) is a key parameter for the liposome membrane electroporation process and hence for the release and oxidation of its redox content during the collision onto UMEs. The presence of ferrocenemethanol as an additional redox probe in the aqueous solution (at room temperature and without addition of surfactant) is also an interesting strategy to detect current spikes corresponding to single redox DMPC liposome collisions with K3Fe(CN)6/K4Fe(CN)6 as the encapsulated aqueous redox probe.

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor.

Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct β-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (kd) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.

Fluorimetrijska analiza permeabilnosti lipozomalnih membrana

The present study provides insight into changes in the liposomal membrane fluidity as a result of variation of structural components (two types of standard phospholipids - 1,2-dipalmitoyl-sn-glycerero3-phosphocholine, i.e. DPPC and 1-palmitoyl-2-oleoylglycerol-3-phosphocholine, i.e. POPC, one type of phospholipid mixture - Lipoid H and sterols - cholesterol and b-sitosterol), as well as variation in sterol concentration (5-50%). Fluorescence spectroscopy (anisotropy, r) has shown that DPPC liposomal membranes were more rigid compared to POPC and Lipoid H lipid bilayers. Furthermore, the increment of sterol concentration has caused a decrease in the membrane fluidity in all liposomal samples. On the other hand, phospholipid bilayers containing b-sitosterol has possessed better fluidity in comparison to their cholesterol parallels. Higher permeability of b-sitosterol liposomal membranes, as well as the beneficial effects of phytosterol on human health, favor the use of b-sitosterol in the preparation of liposomes as components of food, pharmaceutical and cosmetic products.

Evaluation of Hydrodynamic Properties of Bubble Columns Based on Membrane Permeability of Liposomes

Evaluation of Hydrodynamic Properties of Bubble Columns Based on Membrane Permeability of Liposomes

Corticoids modulate liposome membrane fluidity and permeability depending on membrane composition and experimental protocol design

Given that literature data may give inconsistent results on the effect of a drug on lipid membrane properties, this work aims to investigate the impact of the liposome composition and experimental protocol design on glucocorticoids (GRs: cortisol, cortisone, fludrocortisone acetate, methylprednisolone, prednisolone and prednisone)-modulating membrane fluidity and permeability. GRs-loaded liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) were prepared by reverse phase evaporation technique (REV) at DPPC:CHOL:GR molar ratios of 100:100:2.5, and 100:100:10. The formulations were characterized for their size and homogeneity, encapsulation efficiency and loading rates of GRs, incorporation rates and loading rates of DPPC and CHOL. Changes in DPPC membrane fluidity (CHOL% 0, 10, 20, 30 and 100) after exposure to methylprednisolone were monitored by using 5- and 16-doxyl stearic acids (DSA) as spin probes. For permeability studies, the above-mentioned GRs-loaded liposomes and the preformed liposomes exposed to GRs (2.5 mol%) were compared for the leakage of an encapsulated fluorescent dye, sulforhodamine B (SRB), at 37 °C in buffer (pH 7.5) containing NaCl. The SRB release kinetics were analyzed by the Higuchi model for two release phases (from 0 to 10 h, and from 10 to 48 h). All formulations exhibited a monodispersed size distribution of liposomes with a mean particle value close to 0.4 μm, also the DPPC and CHOL were highly incorporated (>95%). High loading rate values of DPPC and CHOL were also obtained. Except for fludrocortisone acetate (51%) and prednisolone (77%), high loading rate values of GRs were obtained (>81%). Fluidity and permeability studies showed that the GR concentration, CHOL content, experimental protocol design including the period of incubation represent critical parameters to be considered in analyzing the effect of drugs on the membrane properties.

Tuning Liposome Membrane Permeability by Competitive Coiled Coil Heterodimerization and Heterodimer Exchange.

Membrane-active peptides that enable the triggered release of liposomal cargo are of great interest for the development of liposome-based drug delivery systems but require peptide-lipid membrane interactions that are highly defined and tunable. To this end, we have explored the possibility to use the competing interactions between membrane partitioning and heterodimerization and the folding of a set of four different de novo designed coiled coil peptides. Covalent conjugation of the cationic peptides triggered rapid destabilization of membrane integrity and the release of encapsulated species. The release was inhibited when introducing complementary peptides as a result of heterodimerization and folding into coiled coils. The degree of inhibition was shown to be dictated by the coiled coil peptide heterodimer dissociation constants, and liposomal release could be reactivated by a heterodimer exchange to render the membrane bound peptide free and thus membrane-active. The possibility to tune the permeability of lipid membranes using highly specific peptide-folding-dependent interactions delineates a new possible approach for the further development of responsive liposome-based drug delivery systems.

1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

Membrane Rigidity Determined by Atomic Force Microscopy Is a Parameter of the Permeability of Liposomal Membranes to the Hydrophilic Compound Calcein.

We determined the permeability coefficient of a model hydrophilic drug, calcein, encapsulated within saturated lipid-based nano-sized liposomes of various lipid profiles. We demonstrated that the addition of cholesterol to liposomes containing saturated lipids increased the permeability of the liposomal membrane to calcein via a decrease in the membrane bending modulus, as determined by means of atomic force microscopy. We found an inverse correlation between the membrane bending modulus of saturated lipid-based nano-sized liposomes and the permeability coefficient of encapsulated calcein, demonstrating that bending modulus, as determined by means of atomic force microscopy, is a quantitative parameter describing the permeability of liposomal membranes to calcein.

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.

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies.

Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity.

Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

PH-Sensitive liposomes with embedded 3,7-diazabicyclo[3.3.1]nonane derivative

1,5-Dinitro-3,7-ditridecyl-3,7-diazabicyclo[3.3.1]nonane modulates the permeability of liposomal membranes as pH of surrounding aqueous solution decreases from 9 down to 5.