DMPC Liposomes Research Articles

The (un)known issue with using rose bengal as a standard of singlet oxygen photoproduction.

Rose bengal (RB) is a widely used photosensitizer for determining quantum yields of singlet oxygen generation. While it is known to aggregate in polar environments at concentrations above 2 μM, the relationship between RB concentration and singlet oxygen photogeneration remains unclear. This study investigates the shift from monomeric to dimeric RB with increasing concentration and its impact on singlet oxygen generation in D2O-based solutions and DMPC liposomes. Absorbance maxima for RB were observed at 514 nm (dimer) and 549 nm (monomer), with ionic environments influencing aggregation rates. Singlet oxygen phosphorescence showed non-linear dependency above 2 μM, indicating the effects of aggregation. Results suggest that RB concentrations should be kept at 1 μM or lower in photochemical studies to avoid aggregation-related discrepancies in singlet oxygen yield determination. These findings highlight the importance of considering RB aggregation in photochemical research and medical applications.

Effect of DMSO on Structural Properties of DMPC and DPPC Liposome Suspensions.

The study and characterization of the biophysical properties of membranes and drug-membrane interactions represent a critical step in drug development, as biological membranes act as a barrier that the drug must overcome to reach its active site. Liposomes are widely used in drug delivery to circumvent the poor aqueous solubility of most drugs, improving systemic bioavailability and pharmacokinetics. Further, they can be targeted to deliver to specific disease sites, thus decreasing drug load, and reducing side effects and poor adherence to treatment. To improve drug solubility during liposome preparation, DMSO is the most widely used solvent. This raises concern about the potential effect of DMSO on membranes and leads us to investigate, using DSC and EPR, the influence of DMSO on the behavior of lipid model membranes of DMPC and DPPC. In addition, we tested the influence of DMSO on drug-membrane interaction, using compounds with different hydrophobicity and varying DMSO content, using the same experimental techniques. Overall, it was found that with up to 10% DMSO, changes in the bilayer fluidity or the thermotropic properties of the studied liposomes were not significant, within the experimental uncertainty. For higher concentrations of DMSO, there is a stabilization of both the gel and the rippled gel phases, and increased bilayer fluidity of DMPC and DPPC liposomes leading to an increase in membrane permeability.

Phospholipid-induced secondary structural changes of lysozyme polymorphic amyloid fibrils studied using vacuum-ultraviolet circular dichroism.

The hallmark of amyloidosis, such as Alzheimer's disease and Parkinson's disease, is the deposition of amyloid fibrils in various internal organs. The onset of the disease is related to the strength of cytotoxicity caused by toxic amyloid species. Furthermore, amyloid fibrils show polymorphism, where some types of fibrils are cytotoxic while others are not. It is thus essential to understand the molecular mechanism of cytotoxicity, part of which is caused by the interaction between amyloid polymorphic fibrils and cell membranes. Here, using amyloid polymorphs of hen egg white lysozyme, which is associated with hereditary systemic amyloidosis, showing different levels of cytotoxicity and liposomes of DMPC and DMPG, changes in the secondary structure of the polymorphs and the structural state of phospholipid membranes caused by the interaction were investigated using vacuum-ultraviolet circular dichroism (VUVCD) and Laurdan fluorescence measurements, respectively. Analysis has shown that the more cytotoxic polymorph increases the antiparallel β-sheet content and causes more disorder in the membrane structure while the other less cytotoxic polymorph shows the opposite structural changes and causes less structural disorder in the membrane. These results suggest a close correlation between the structural properties of amyloid fibrils and the degree of structural disorder of phospholipid membranes, both of which are involved in the fundamental process leading to amyloid cytotoxicity.

Initial Quenching Efficiency Determines Light-Driven H2 Evolution of [Mo3 S13 ]2- in Lipid Bilayers.

Nature uses reactive components embedded in biological membranes to perform light-driven photosynthesis. Here, a model artificial photosynthetic system for light-driven hydrogen (H2 ) evolution is reported. The system is based on liposomes where amphiphilic ruthenium trisbipyridine based photosensitizer (RuC9 ) and the H2 evolution reaction (HER) catalyst [Mo3 S13 ]2- are embedded in biomimetic phospholipid membranes. When DMPC was used as the main lipid of these light-active liposomes, increased catalytic activity (TONCAT ~200) was observed compared to purely aqueous conditions. Although all tested lipid matrixes, including DMPC, DOPG, DPPC and DOPG liposomes provided similar liposomal structures according to TEM analysis, only DMPC yielded high H2 amounts. In situ scanning electrochemical microscopy (SECM) measurements using Pd microsensors revealed an induction period of around 26 minutes prior to H2 evolution, indicating an activation mechanism which might be induced by the fluid-gel phase transition of DMPC at room temperature. Stern-Volmer-type quenching studies revealed that electron transfer dynamics from the excited state photosensitizer are most efficient in the DMPC lipid environment giving insight for design of artificial photosynthetic systems using lipid bilayer membranes.

Design of light-responsive amphiphilic self-assemblies: A novel application of the photosensitive diazirine moiety

Diazirine is one of the smallest photo-sensitive moieties discovered to date. When incorporated in the structure of phospholipids, its minimal size has a low impact on the morphology of the resultant liposomes. A DMPC-diazirine analogue was designed and subsequently used to generate liposomes with a lower permeability and a lower phase-transition temperature compared to control DMPC liposomes. Contrary to control liposomes, in the absence of light, the photosensitive nanoparticles retained the cargo (calcein) for at least 10 days. However, upon irradiation, diazirine’s conversion triggered the fluorophore release within minutes. The kinetics of the release could be tuned by the power and duration of the irradiation process. The same approach can be used on other nanomaterials, with the final goal of discovering a release profile appropriate not only for therapeutic applications, but also for agrochemicals delivery or cosmoceutics.

Sulfonated polystyrenes: pH and Mg2+-insensitive amphiphilic copolymers for detergent-free membrane protein isolation

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

Surfactant-regulated acetylpyrene assemblies as fluorescent probes for identifying heme proteins in an aqueous solution

Heme proteins play various important roles in a variety of physiological and pathological processes. Surfactant assemblies have drawn great attention in fabricating fluorescent sensors to detect and identify proteins. In this study, an acetylpyrene fluorophore containing imidazole HP-1 was synthesized, and it could be well modulated by an anionic surfactant sodium dodecyl sulfate (SDS). The selected ensemble based on HP-1/SDS assemblies exhibited selective fluorescence sensing performance towards the heme proteins, including neuroglobin (Ngb), myoglobin (Mb) and cytochrome c (Cyt c). Besides, phospholipid DMPC vesicles as membrane models were particularly explored the association process between the heme protein Mb and membrane. The present work showed that Mb induced the lysis of DMPC liposomes visualized by transmission electron microscopy and optical microscope.

Stability of DMPC Liposomes Externally Conjugated with Branched Polyglycerol.

Vesicles formed by DMPC liposomes externally conjugated with branched polyglycerol-dendrons as well as linear PEG in water solution were simulated using the DPD method. Such a structure of vesicles corresponds to the structure of polymer-grafted liposomes obtained experimentally by the post-insertion method, in which polymer chains are fixed on the outer surface of the liposome. The grafting density, generation number and spacer length of grafted dendrons were varied. It was shown that modification of the outer surface of liposomes due to grafting of hydrophilic dendrons has practically no effect on the size and shape of the vesicle, as well as on the morphology of the lipid membrane up to certain critical thresholds of grafting density, degree of polymerization, and generation number of grafted molecules. Exceeding the threshold values of these structural parameters leads to irreversible deformation of the lipid membrane. Diffusion through the membrane and the transition of grafted molecules from the outer surface of the liposome to the inner surface is not observed for dendrons with a generation number higher than one, even at high grafting densities. The critical values of the generation number and the characteristics of the molecular coating at these values were determined for various grafting densities and spacer lengths of the grafted chains. It was shown that the chemical potential of the grafted dendron can serve as a stability metric for the conjugated liposome. The chemical potential of grafted molecules was calculated using the mean field model of the spherical brush on the liposome surface. An analysis of the simulation data shows that, within the framework of the applicability of the mean field approach, the value of the chemical potential is a sufficient criterion for separating vesicles into stable and unstable forms. These results can be used as a guide for the experimental design of nanocontainers based on lipid vesicles with an external protective coating of branched macromolecules.

(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

Liposome-based nanocapsules for the controlled release of dietary curcumin: PDDA and silica nanoparticle-coated DMPC liposomes enhance the fluorescence efficiency and anticancer activity of curcumin.

Nanosystems with various compositions and biological properties are being extensively investigated for drug and gene delivery applications. Many nanotechnology methods use novel nanocarriers, such as liposomes, in therapeutically targeted drug delivery systems. However, liposome matrices suffer from several limitations, including drug leakage and instability. Therefore, the surface modification of liposomes by coating them or adding polymers has advanced their application in drug delivery. Hence, the prevention of drug release from the liposome bilayers was the main focus of this work. For this purpose, liposomes were synthesized according to a thin film hydration method by applying various surface modifications. Three different nanocapsules, N1, N2, and N3, were prepared using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), poly(diallyldimethylammonium)chloride (PDAA) polymer, and silica nanoparticles. PDDA and silica nanoparticles were coated on the surface of liposomes using a layer-by-layer assembly method, completely encapsulating curcumin into the core of the liposome. Fluorescence spectroscopy, TGA, DLS, XRD, SEM, and zeta potential methods were used to characterize the prepared nanocapsules. Interestingly, the fluorescence of curcumin showed a blue shift and the fluorescence efficiency was extraordinarily enhanced ∼25-, ∼54-, and ∼62-fold in the N1, N2, and N3 nanocapsules, respectively. Similarly, encapsulation efficiency, drug loading, and the anticancer activity of dietary curcumin were investigated for the different types of DMPC nanocapsules. The drug efficiencies of the liposomes were established according to the release of curcumin from the liposomes. The results showed that the release of curcumin from the nanocapsules decreased as the number of layers at the surface of the liposomes increased. The release of curcumin follows the Higuchi model; thus, a slow rate of diffusion is observed when a number of layers is added. The better encapsulation and higher anti-cancer activity of curcumin were also observed when more layers were added, which is due to electrostatic interactions inhibiting curcumin from being released.

Outstanding Enhancement of Curcumin Fluorescence in PDDA and Silica Nanoparticles Coated DMPC Liposomes Based Nanocapsules: Application for Selective Estimation of ATP**

AbstractHere we illustrate the synthesis of, 2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) liposome based poly(dimethyldiallylammonium chloride) (PDDA) nanocapsule where PDDA and silica nanoparticles are coated on the surface of curcumin embedded liposomes. The prepared nanocapsule was analyzed using UV‐vis spectroscopy, fluorescence spectroscopy, Dynamic Light Scattering technique, Scanning Electron Microscopy, Zeta‐potential, Thermogravimetric analysis and X‐ray diffraction method. The fluorescence intensity of DMPC nanocapsules was found to be exceedingly, 60‐fold, higher than free curcumin, thus, it boosts potential of such nanocapsules for various applications. The detection of Adinosine Triphosphate (ATP) was carried out by monitoring the change in the fluorescence emission of this DMPC nanocapsules while increasing the concentration of ATP molecule in the tested sample. Interestingly, fluorescence of free curcumin did not respond to ATP, whereas the fluorescence intensity of nanocapsules increased by the increase of ATP concentration, which establishes that materials prepared using nanotechnological modification of probe molecule can selectively target a particular analytical species. The nanocapsules based probe gave a linear correlation between the fluorescence intensity of DMPC nanocapsules in the concentration range 1 μM −100 μM with the limit of detection equals to 0.5 μM. The described method is not interfered by uracil triphosphate, guanine triphosphate, cytosine triphosphate and thymine triphosphate molecules and showed good stability.

Selective prototropism of lumichrome in the liposome/graphene oxide interface: A detailed spectroscopic study

The interactions of the biologically active fluorophores like flavins and biomolecules with nano carriers are very much important to produce targeted drug delivery systems. Also studying the interaction of graphene oxide (GO) with liposomes is essential for surface science and which have various applications. Herein, we have reported the interaction of a biologically active flavin molecule lumichrome (LU) with GO, in the absence and presence of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome by employing different spectroscopic techniques. These study also shows the prototropic tautomerization of LU, when it interacted with GO in the absence and presence of DMPC liposome. We have found that GO interacted with LU differently in the absence and presence of DMPC liposome. In the absence of DMPC liposome, LU molecules were directly adsorbed on the GO surface involving both edges and basal planes of GO via hydrogen bonding, π-π stacking between aromatic ring of LU and GO, and as a result we observe higher fluorescence quenching of LU. However, in the presence of DMPC liposome, LU molecules were partitioned inside the vesicle and in the presence of GO whole vesicle (in which LU molecules were partitioned) was placed on the edges of GO and here π-π stacking between aromatic ring of LU and GO is not possible, and as a result we observe moderate fluorescence quenching of LU. This study also indicates that zwitterionic DMPC liposome retained its structure when it interacts with GO. We have also found that in LU + GO complex, due to the direct adsorption of LU in GO surface, the prototropic tautomerization of LU is not possible, only the alloxazine form of LU exist. However, in LU + DMPC + GO complex when LU is incorporate inside the vesicle both alloxazine and isoalloxazine form of LU co-exist, indicates the prototropic tautomerization of LU in LU + DMPC + GO complex.

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

Method to Improve Azo-Compound (AAPH)-Induced Hemolysis of Erythrocytes for Assessing Antioxidant Activity of Lipophilic Compounds

We examined the method of oxidative hemolysis for assessment of antioxidant activity of various compounds, especially lipophilic compounds. 2,2'-Azobis(amidinopropane) dihydrochloride (AAPH) was used as the source of free radicals for the oxidative hemolysis of horse erythrocytes. We found that absorbance at 540 nm is not appropriate for monitoring AAPH-induced hemolysis. Instead, we should use absorbance at 523 nm (an isosbestic point), because AAPH oxidizes the oxygenated hemoglobin to methemoglobin and absorbance at 540 nm does not correctly reflect the amount of released hemoglobin by AAPH-induced hemolysis. The corrected method of AAPH-induced hemolysis was applicable to assess the antioxidant activity of various hydrophilic compounds such as ascorbic acid, (-)-epicatechin, and edaravone. For the assessment of antioxidant activity of lipophilic compounds, we need appropriate dispersing agents for these lipophilic compounds. Among several agents tested, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) liposome at a concentration of 0.34 mM was found to be useful. Exogenous α-tocopherol incorporated using DMPC liposome as a dispersing agent was shown to protect erythrocytes from AAPH-induced hemolysis in a concentration-dependent manner.

Trehalose liposomes induce apoptosis of breast tumor cells in vitro and in vivo

The inhibitory effects of trehalose liposomes (TL) comprising l-α-dimyristoylphosphatidylcholine (DMPC) and α-D-glucopyranosyl-α-D-glucopyranoside monomyritate (TreC14) were investigated on breast cancer MDA-MB-453 cells in vitro and in vivo. The IC50 values of TL for MDA-MB-453 cells were remarkably lower than those of DMPC liposomes. The inhibitory effects of TL on the proliferation of MDA-MB-453 cells mediated via apoptosis induction were observed following their accumulation on MDA-MB-453 cell membranes. The membrane fluidity of MDA-MB-453 cells increased after TL treatment, as evident from a fluorescence depolarization assay. TL induced the apoptosis of MDA-MB-453 cells through caspase activation and mitochondrial membrane potential reduction, and suppressed the nuclear factor kappa B activity. A remarkable reduction in tumor volume was observed in a human breast cancer mouse model topically treated with TL. Induction of apoptosis was evident in TL-treated breast cancer tumors of mice using the TUNEL assay.

Unravelling the mechanism of action of “de novo” designed peptide P1 with model membranes and gram-positive and gram-negative bacteria

In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the “de novo” designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium.All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.

The acid\u2013base and redox properties of menaquinone MK-4, MK-7, and MK-9 (vitamin K2) in DMPC monolayers on mercury

The acid–base and redox properties of the menaquinones MK-4, MK-7, and MK-9 (vitamin K2) have been studied in DMPC monolayers on mercury electrodes. The monolayers were prepared by adhesion-spreading of menaquinone-spiked DMPC liposomes on a stationary mercury drop electrode. All three menaquinones possess {text{p}}K_{{text{a}}} constants outside the experimentally accessible range, i.e., they are higher than about 12. The standard potentials of MK-4, MK-7, and MK-9 in the DMPC monolayers are very similar, i.e., 0.351, 0.326, and 0.330 V (corresponding to the biochemical standard potentials − 0.063, − 0.088, and − 0.085 V).Graphic abstract

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.

Antioxidant Activity of Metal Nanoparticles Coated with Tocopherol-Like Residues-The Importance of Studies in Homo- and Heterogeneous Systems.

Functionalized nanoparticles (NPs) attract great attention in pharmacy, diagnostics, and biomedical areas due to benefits like localization and unique interactions of NPs with biocomponents of living cells. In the present paper, we prepared and characterized two kinds of gold nanoparticles (AuNPs) coated with α-tocopherol-like residues: 1A were soluble in non-polar solvents and their antioxidant activity was tested during the peroxidation of a model hydrocarbon in a homogeneous system, whereas nanoparticles 1B were soluble in polar solvents and were applied as antioxidants in micellar and liposomal systems. The effectiveness of 1A is comparable to 2,2,5,7,8-pentamethylchroman-6-ol (PMHC, an analogue of α-tocopherol). Taking the results of the kinetic measurements, we calculated an average number of 2150 chromanol residues per one NP, suggesting a thick organic coating around the metal core. In heterogeneous systems, the peroxidation of methyl linoleate dispersed in Triton X-100 micelles or DMPC liposomes resulted in the observation that 1B (545 chromanol residues per one NP) was active enough to effectively inhibit peroxidation in a micellar system, but in a liposomal system, 1B behaved as a retardant (no clear induction period). The importance of microenvironment in heterogeneous systems on the overall antioxidant activity of nanoparticles is discussed.

Investigation of Different Prototropic Forms of Biologically Active Flavin Lumichrome in the Presence of Liposome.

Herein, we reported the photophysical behavior of lumichrome (LC), one of the biologically active flavin molecules, in the presence of small unilamellar vesicle of anionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). With the help of different spectroscopic techniques, we have proposed that anionic DMPC liposome interacts with the cationic form LC in ground state and in excited state and modulate the spectral properties of LC. Photophysical study reveals that different prototropic forms of LC are present in DMPC liposome medium. In the presence of DMPC liposome, fluorescence emission properties of LC vary with change in excitation and emission wavelengths. This indicates switch over between different structural forms of LC. From fluorescence lifetime measurements and fluorescence lifetime imaging (FLIM) study, it was revealed that emission decay profile of LC was fitted biexponentially in the presence of liposome. It suggests that in the presence of liposome, more than one form of LC is present. We have constructed the time-resolved area-normalized emission spectra (TRANES) of LC in the liposome and found one isoemissive point. This confirmed that two emissive species of LC are present in liposome. FLIM study and FE-SEM study give an idea about the structural feature of the complex between LC and liposome.