Transmembrane pH Gradient Research Articles

Lyoprotective Effects of Mannitol and Lactose Compared to Sucrose and Trehalose: Sildenafil Citrate Liposomes as a Case Study.

The lyoprotective effects of mannitol and lactose have been evaluated in the production of sildenafil citrate liposomes. Liposomes were prepared by mixing the components under ultrasonic agitation, followed by a transmembrane pH gradient for remote drug loading. Mannitol and lactose, as compared to sucrose and trehalose, were used as the stabilizing agents, and different freeze-drying cycles were assayed. The remaining moisture and the thermal characteristics of the lyophilized samples were analyzed. Size, entrapment efficiency, biocompatibility, and cell internalization of original and rehydrated liposomes were compared. The type of additive did not affect the biocompatibility or cell internalization, but did influence other liposome attributes, including the thermal characteristics and the remaining moisture of the lyophilized samples. A cut-off of 5% (w/w) remaining moisture was an indicator of primary drying completion—information useful for scaling up and transfer from laboratory to large-scale production. Lactose increased the glass transition temperature to over 70 °C, producing lyoprotective effects similar to those obtained with sucrose. Based on these results, formulations containing liposomes lyophilized with lactose meet the FDA’s requirements and can be used as a biocompatible and biodegradable vehicle for the pulmonary delivery of therapeutic doses of sildenafil citrate.

Cholesterol Levels Affect the Performance of AuNPs-Decorated Thermo-Sensitive Liposomes as Nanocarriers for Controlled Doxorubicin Delivery

Stimulus-responsive liposomes (L) for triggering drug release to the target site are particularly useful in cancer therapy. This research was focused on the evaluation of the effects of cholesterol levels in the performance of gold nanoparticles (AuNPs)-functionalized L for controlled doxorubicin (D) delivery. Their interfacial and morphological properties, drug release behavior against temperature changes and cytotoxic activity against breast and ovarian cancer cells were studied. Langmuir isotherms were performed to identify the most stable combination of lipid components. Two mole fractions of cholesterol (3.35 mol% and 40 mol%, L1 and L2 series, respectively) were evaluated. Thin-film hydration and transmembrane pH-gradient methods were used for preparing the L and for D loading, respectively. The cationic surface of L allowed the anchoring of negatively charged AuNPs by electrostatic interactions, even inducing a shift in the zeta potential of the L2 series. L exhibited nanometric sizes and spherical shape. The higher the proportion of cholesterol, the higher the drug loading. D was released in a controlled manner by diffusion-controlled mechanisms, and the proportions of cholesterol and temperature of release media influenced its release profiles. D-encapsulated L preserved its antiproliferative activity against cancer cells. The developed liposomal formulations exhibit promising properties for cancer treatment and potential for hyperthermia therapy.

Biochemical and biophysical pH clamp controlling Net H+ efflux across the plasma membrane of plant cells.

P-type H+ ATPases mediate active H+ efflux from plant cells. They generate a proton motive force across the plasma membrane, providing the free energy to drive the transport of other solutes, partly by coupling to H+ influx. Wegner & Shabala (2020) recently suggested that passive H+ influx can exceed pump-driven efflux due to 'active buffering', that is, cytosolic H+ scavenging and apoplastic H+ generation by metabolism ('biochemical pH clamp'). Charge balance is provided by K+ efflux or anion influx. Here, this hypothesis is extended to net H+ efflux: even though H+ pumping is faster than backflow via symporters and antiporters, a progressive increase in the transmembrane pH gradient is avoided. Cytosolic H+ release is associated with bicarbonate formation from CO2 . Bicarbonate serves as substrate for the PEPCase, catalyzing the reaction from phosphoenolpyruvate to oxaloacetate, which is subsequently reduced to malate. Organic anions such as malate and citrate are released across the plasma membrane and are (partly) protonated in the apoplast, thus limiting pump-induced acidification. Moreover, a 'biophysical pH clamp' is introduced, that is, adjustment of apoplastic/cytosolic pH involving net H+ fluxes across the plasma membrane, while the gradient between compartments is maintained. The clamps are not mutually exclusive but are likely to coexist.

The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC50 of 2 µM, and TbAQP3 to a much lesser extent (IC50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

Purification and antimicrobial mechanism of a novel bacteriocin produced by Lactobacillus rhamnosus 1.0320

For a long time, the abuse of antibiotics and the excessive use of synthetic preservatives have caused harm to human health. Bacteriocins produced by lactic acid bacteria (LAB) are nontoxic and exhibit potential as natural preservatives. In this experiment, the bacteriocin 1.0320 produced by Lactobacillus rhamnosus 1.0320 screened from traditional koumiss was used as the research object. The main purpose of the experiment was to purify bacteriocin 1.0320 and study its antimicrobial mechanism. Bacteriocin 1.0320 exhibited antimicrobial activity against gram-positive and gram-negative bacteria. Bacteriocin 1.0320 in cell-free fermentation supernatant (CFS) was purified by ethyl acetate extraction and anion exchange chromatography. The molecular mass of bacteriocin 1.0320 was 1–3.3 KDa as assessed by dialysis and Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). The minimum inhibitory concentration of bacteriocin 1.0320 on Escherichiacoli UB1005 was 0.072 mg/mL. Bacteriocin 1.0320 can form pores on the surface of cell membrane, increase the permeability of cell membrane, dissipate the cytoplasmic membrane potential and transmembrane pH gradient, and destroy the integrity of cell membrane, causing cell content to flow out and cell death. The effect is related to the concentration of bacteriocin 1.0320. This information provides the theoretical basis for the antimicrobial mechanism of L. rhamnosus bacteriocin.

Synthesis and characterization of turmeric oil loaded non-ionic surfactant vesicles (niosomes) and its enhanced larvicidal activity against mosquito vectors

The fabrication of smart materials and its potential versatility instigated to develop Niosomes for sustained delivery of drugs. In this pursuit, in this research work, we have synthesized Niosomes using the transmembrane pH gradient method from turmeric oil and evaluated against mosquito vectors. The characteristics of niosomes were extensively explored by Fourier Transform Infra-Red Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Zeta Potential and others. FTIR analysis affirmed the formation of Niosomes from turmeric oils. Subsequently, the synthesized niosomes were observed in SEM to decipher the size and shape. DLS and Zeta Potential studies indicated that synthesized niosomes average particle size was around 491.09 nm with good stability. Functionally, the synthesized Niosomes were evaluated against third instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi . The outcome of the study demonstrated that niosomes exhibit effective mortality against the larvae in a dose-dependent method with significant Lethal Concentration (LC) values. Further, histopathological analysis of treated larvae implies that niosomes cause extensive damage in the midgut region and the collapse of internal membrane bodies. Overall, the comprehensive study of the synthesized niosomes from turmeric oil will be competent for the vector management program.

KV11.1 Potassium Channel and the Na+/H+ Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells.

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H+/HCO3- transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K+ channels, which are central regulators of cell membrane potential (Vm), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and KV11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na+/H+ antiporter NHE1. Blocking KV11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pHi increase. Interestingly, both NHE1 and KV11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, KV11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between KV11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.

Synthesis of carboxymethylated β-glucan from naked barley bran and its antibacterial activity and mechanism against Staphylococcus aureus

This study aimed to elucidate the antimicrobial efficacy of carboxymethylated β-glucan (CMG) against Staphylococcus aureus and the possible underlying mechanisms. β-Glucan (BG) was extracted from naked barley bran, then ultrafiltrated to obtain four fractions (BG-1, BG-2, BG-3, BG-4). Five types of CMG were prepared using an amidation reaction and characterized by FTIR spectroscopy. The underlying mechanisms were studied using changes in the transmembrane electrical potential, pH gradient, and intracellular ATP, confocal laser scanning microscopy, and electron microscopy. The data showed that CMG-2 had the highest antibacterial activity. CMG-2 induced membrane permeabilization, morphological changes, and structural disruption in S. aureus cells, with the formation of blebs and microspheres, the disruption of cell walls and membranes, and cell lysis. Our findings provide insight into the antimicrobial mechanism of CMG against S. aureus, which will provide a foundation for developing CMG as a novel antibacterial agent in the food and biomedical fields.

Anti-Salmonella mode of action of natural L-phenyl lactic acid purified from Lactobacillus plantarum ZJ316.

Salmonella is a serious foodborne pathogen responsible for more than 90 million cases of gastroenteritis worldwide annually. Due to the gradual increase in antibiotic-resistant Salmonella strains, the identification of natural antibacterial substances is urgently needed. Herein, we purified natural L-phenyl lactic acid (L-PLA) from Lactobacillus plantarum ZJ316 and revealed its antimicrobial mode against Salmonella enterica subsp. enterica ATCC 14028. L-PLA (98.14% pure) was obtained using the macroporous resin XAD-16, solid-phase extraction (SPE), reverse-phase high-performance liquid chromatography (RP-HPLC), and chiral chromatography. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results showed that the cell morphology was severely compromised. Transmembrane electrical potential (ΔΨ), transmembrane pH gradient (ΔpH), intracellular ATP level, extracellular electrical conductivity (EC), and genomic DNA analyses were employed to evaluate the antibacterial mode of action of L-PLA. The proton motive force (PMF) and ATP of Salmonella cells rapidly dissipated, and the EC markedly increased. The gel retardation assay demonstrated that L-PLA could bind to genomic DNA and intercalate into the nucleic acids. The anti-Salmonella mode of action of L-PLA was attributed to the destruction of the cell membrane and genomic DNA binding. This research suggests that L-PLA has potential applications as an antimicrobial agent in food, medicine, and other fields. KEY POINTS: • Natural L-PLA was purified from L. plantarum ZJ316 with a purity of 98.14%. • L-PLA effectively inhibited Salmonella strains by antibacterial activities and MICs. • Membrane destruction and binding with DNA are the anti-Salmonella modes of L-PLA.

Engineered Polymersomes for the Treatment of Fish Odor Syndrome: A First Randomized Double Blind Olfactory Study.

Trimethylamine (TMA) is a metabolite overtly present in patients suffering from trimethylaminuria (TMAU), a rare genetic disorder characterized by a strong “fishy” body odor. To date, no approved pharmacological treatment to sequester excess TMA on the skin of patients exists. Here, transmembrane pH gradient poly(isoprene)‐block‐poly(ethylene glycol) (PI‐b‐PEG) polymersomes are investigated for the topical removal of TMA. PI‐b‐PEG amphiphiles of varying chain length are synthesized and evaluated for their ability to form vesicular structures in aqueous media. The optimization of the PI/PEG ratio of transmembrane pH gradient polymersomes allows for the rapid and efficient capture of TMA both in solution and after incorporation into a topical hydrogel matrix at the pH of the skin. A subsequent double blind olfactory study reveals a significant decrease in perceived odor intensity after application of the polymersome‐based formulation on artificial skin substrates that has been incubated in TMA‐containing medium. This simple and novel approach has the potential to ease the burden of people suffering from TMAU.

Formulation and Evaluation of Mupirocin Nimosomal Gel for Topical Drug Delivery System

The present study was to formulate and evaluate the mupirocin niosomal gel using surfactants span 80 & tween 80 for the preparation of niosomes. Mupirocin entrapped niosomes were prepared by ether injection method and transmembrane pH gradient drug uptake process. Niosomes were prepared by altering the ratios between various non-ionic surfactants (span 80 & tween 80) whereas the concentration of cholesterol and drug was kept constant. The prepared niosomes were characterized for size, shape, entrapment efficiency, invitro drug release studies. The highest entrapment efficiency(99.17%) and drug release (96.14%) was obtained for tween 80 (1:5:5) prepared by transmembrane pH gradient method. The best formulation among the two techniques was selected for incorporated into gel formulation. The prepared niosomal gel and plain gel were subjected to evaluation studies like drug content, invitro drug diffusion studies. The studies were demonstrated that niosomal gel was shown beter pharmacological activity than the conventional mupirocin gel. Based on the results it was concluded that niosomal preparations offers more advantageous than the conventional preparations.

A Model Protometabolic Pathway Across Protocell Membranes Assisted by Photocatalytic Minerals.

Protocell analogs (lipid vesicles) to modern cell membranes have been postulated as compartments that may have been involved in primordial metabolism during the transition from geochemistry to biochemistry on early Earth. The transduction of light energy into chemical energy for metabolism was a key step in the transition from the earliest metabolisms to phototrophy. Photocatalytic minerals may have served the role of enzymes during these transitional stages. Here, we demonstrate a simple photoheterotrophic protometabolism promoted by photocatalytic minerals across a model protocell (vesicle) membrane. These minerals in the extra-vesicular medium utilized light energy to drive a coupled, multi-step transmembrane electron transfer reaction (TMETR), while simultaneously generating a transmembrane pH gradient and reducing nicotinamide adenine dinucleotide (NAD+) to NADH within the vesicle. The proton gradient or chemiosmotic potential could have provided a basis for adenosine triphosphate (ATP) synthesis and NADH could potentially have driven further metabolic chemistry inside the protocells.

A Model Protometabolic Pathway across Protocell Membranes Assisted by Photocatalytic Minerals

Protocell analogs (lipid vesicles) to modern cell membranes have been postulated as compartments that may have been involved in primordial metabolism during the transition from geochemistry to biochemistry on early Earth. The transduction of light energy into chemical energy for metabolism was a key step in the transition from the earliest metabolisms to phototrophy. Photocatalytic minerals may have served the role of enzymes during these transitional stages. Here, we demonstrate a simple photoheterotrophic protometabolism promoted by photocatalytic minerals across a model protocell (vesicle) membrane. These minerals in the extra-vesicular medium utilized light energy to drive a coupled, multistep transmembrane electron transfer reaction (TMETR), while simultaneously generating a transmembrane pH gradient and reducing nicotinamide adenine dinucleotide (NAD+) to NADH within the vesicle. The proton gradient or chemiosmotic potential could have provided a basis for adenosine triphosphate (ATP) synthesis and ...

Dual Loading Of Primaquine And Chloroquine Into Liposome

Abstract Primaquine (PQ) has long been recognized as the only effective drug in the treatment of hepatic stage malaria. However, severe toxicity limits its therapeutical application. Combining PQ with chloroquine (CQ) has been reported as enhancing the former’s efficacy, while simultaneously reducing its toxicity. In this study, the optimal conditions for encapsulating PQ-CQ in liposome, including incubation time, temperature and drug to lipid ratio, were identified. Furthermore, the effect of the loading combination of these two drugs on liposomal characteristics and the drug released from liposome was evaluated. Liposome is composed of HSPC, cholesterol and DSPE-mPEG2000 at a molar ratio of 55:40:5 and the drugs were loaded by means of the transmembrane pH gradient method. The particle size, ζ-potential and drug encapsulation efficiency were subsequently evaluated. The results showed that all liposome was produced with a similar particle size and ζ -potential. PQ and CQ could be optimally loaded into liposome by incubating the mixtures at 60°C for 20 minutes at a respective drug to lipid ratio of 1:3 for PQ and CQ. However, compared to single drug loading, dual-loading of PQ+CQ into liposome resulted in lower drug encapsulation and slower drug release. In conclusion, PQ and CQ can be jointly loaded into liposome with differing profiles of encapsulation and drug release.

Proton channel blockers inhibit Duox activity independent of Hv1 effects

The NADPH oxidase reaction produces protons. In the case of the NADPH oxidase, NOX2, activity depends on secretion of these protons and is inhibited by blockade of the voltage-gated proton channel (Hv1). Duox1 and Duox2 activities similarly produce intracellular protons but synthesize hydrogen peroxide directly instead of superoxide. Hv1 contributes to acid secretion in some epithelia that express Duox. To test the hypothesis that Duox activity is also sensitive to Hv1 channel blockers, Duox was assayed in the presence of either Zn2+ or 5-chloro-2-guanidinobenzimidazole (ClGBI). Both compounds inhibited Duox activity in normal human bronchial epithelial cells but with an IC50 over 10-fold higher than that reported for Hv1 (IC50 Zn2+ = 0.68 mM; IC50 ClGBI = 0.07–0.14 mM). Homogenized HEK293T cells expressing either Duox1 or Duox2 showed similar IC50 values for ClGBI suggesting these compounds inhibit the enzymes through alternate mechanisms independent of Hv1 proton secretion. Inclusion of superoxide dismutase did not restore Duox hydrogen peroxide synthesis. Addition of nigericin to eliminate any possible transmembrane pH gradients in intracellular membrane-localized Duox did not alter activity in HEK293T homogenates. Extracellular Zn2+ blocked intracellular Ca2+ increases needed for Duox activity. Together the data suggest that Duox enzyme activities in epithelia are inhibited by compounds that block Hv1 but inhibition occurs through Hv1-independent mechanisms and support the idea that Hv1 is not required for Duox activity.

Modeling the Distribution of Diprotic Basic Drugs in Liposomal Systems: Perspectives on Malaria Nanotherapy.

Understanding how polyprotic compounds distribute within liposome (LP) suspensions is of major importance to design effective drug delivery strategies. Advances in this research field led to the definition of LP-based active drug encapsulation methods driven by transmembrane pH gradients with evidenced efficacy in the management of cancer and infectious diseases. An accurate modeling of membrane-solution drug partitioning is also fundamental when designing drug delivery systems for poorly endocytic cells, such as red blood cells (RBCs), in which the delivered payloads rely mostly on the passive diffusion of drug molecules across the cell membrane. Several experimental models have been proposed so far to predict the partitioning of polyprotic basic/acid drugs in artificial membranes. Nevertheless, the definition of a model in which the membrane-solution partitioning of each individual drug microspecies is studied relative to each other is still a topic of ongoing research. We present here a novel experimental approach based on mathematical modeling of drug encapsulation efficiency (EE) data in liposomal systems by which microspecies-specific partition coefficients are reported as a function of pH and phospholipid compositions replicating the RBC membrane in a simple and highly translatable manner. This approach has been applied to the study of several diprotic basic antimalarials of major clinical importance (quinine, primaquine, tafenoquine, quinacrine, and chloroquine) describing their respective microspecies distribution in phosphatidylcholine-LP suspensions. Estimated EE data according to the model described here closely fitted experimental values with no significant differences obtained in 75% of all pH/lipid composition-dependent conditions assayed. Additional applications studied include modeling drug EE in LPs in response to transmembrane pH gradients and lipid bilayer asymmetric charge, conditions of potential interest reflected in our previously reported RBC-targeted antimalarial nanotherapeutics.

Fatty Acid Fueled Transmembrane Chloride Transport.

Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Herewe report a proof-of-concept prototype transmembrane pumping systemin liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel.Based on our findings from dissecting the H+/Cl-flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.

Development of Liposomal Gemcitabine with High Drug Loading Capacity.

Liposomes are widely used for systemic delivery of chemotherapeutic agents to reduce their nonspecific side effects. Gemcitabine (Gem) makes a great candidate for liposomal encapsulation due to the short half-life and nonspecific side effects; however, it has been difficult to achieve liposomal Gem with high drug loading capacity. Remote loading, which uses a transmembrane pH gradient to induce an influx of drug and locks the drug in the core as a sulfate complex, does not serve Gem as efficiently as doxorubicin (Dox) due to the low p Ka value of Gem. Existing studies have attempted to improve Gem loading capacity in liposomes by employing lipophilic Gem derivatives or creating a high-concentration gradient for active loading into the hydrophilic cores (small volume loading). In this study, we combine the remote loading approach and small volume loading or hypertonic loading, a new approach to induce the influx of Gem into the preformed liposomes by high osmotic pressure, to achieve a Gem loading capacity of 9.4-10.3 wt % in contrast to 0.14-3.8 wt % of the conventional methods. Liposomal Gem showed a good stability during storage, sustained-release over 120 h in vitro, enhanced cellular uptake, and improved cytotoxicity as compared to free Gem. Liposomal Gem showed a synergistic effect with liposomal Dox on Huh7 hepatocellular carcinoma cells. A mixture of liposomal Gem and liposomal Dox delivered both drugs to the tumor more efficiently than a free drug mixture and showed a relatively good anti-tumor effect in a xenograft model of hepatocellular carcinoma. This study shows that bioactive liposomal Gem with high drug loading capacity can be produced by remote loading combined with additional approaches to increase drug influx into the liposomes.

Sildenafil Citrate Liposomes for Pulmonary Delivery by Ultrasonic Nebulization

Technological advances in lipid vesicles facilitate optimization of their properties to achieve therapeutic goals and promote alternative drug administration routes. Sildenafil citrate (SC) is orally administered for the treatment of pulmonary hypertension, but local release would be advantageous in terms of efficacy and safety. In the present study, liposomes from egg phosphatidylcholine and cholesterol loaded with SC, with and without d-α-tocopheryl polyethylene glycol 1000 succinate (Vit E TPGS), were prepared by sonication of the components. A transmembrane pH gradient was applied for active loading of liposomes, and the size, zeta potential, and entrapment efficiency (EE%) were determined. The liposomes were lyophilized and then nebulized. The nebulized samples were collected and the EE% was determined. The transmembrane pH gradient produced a significant increase in the EE% (from 17.68 ± 4.25% to 89.77 ± 7.64%) and, after lyophilization, the EE% remained the same as that of the originals, but the size and zeta potential were modified. EE% of liposomes decreased upon nebulization, particularly for those with Vit E TPGS. Thus, the additives used for lyoprotection reduced the impact of nebulization. Additional studies are essential, but according to these results, SC-loaded liposomes can be considered as suitable and safe carriers for the local release of sildenafil in the pulmonary system.

Ionophoric effects of the antitubercular drug bedaquiline.

Bedaquiline (BDQ), an inhibitor of the mycobacterial F1Fo-ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics. In this communication, we demonstrate that BDQ can inhibit ATP synthesis in Escherichia coli by functioning as a H+/K+ ionophore, causing transmembrane pH and potassium gradients to be equilibrated. Despite the apparent lack of a BDQ-binding site, incorporating the E. coli Fo subunit into liposomes enhanced the ionophoric activity of BDQ. We discuss the possibility that localization of BDQ at F1Fo-ATP synthases enables BDQ to create an uncoupled microenvironment, by antiporting H+/K+ Ionophoric properties may be desirable in high-affinity antimicrobials targeting integral membrane proteins.