RESUMO
The impact of stereochemical purity of lipids on their self-assembly behavior is critical for establishing their true phase behavior from their commercial counterparts, which often contains stereoisomeric mixtures and other impurities. Here, stereochemically pure phytantriol (PT), (3,7,11,15-tetramethylhexadecane-1,2,3-triol) was synthesized from the natural trans-phytol and its thermotropic and lyotropic phase behavior in water investigated by small-angle X-ray scattering (SAXS), polarized optical microscopy (POM), and differential scanning calorimetry (DSC). These chemically pure lipids contain two chiral centers at the hydrophilic head group region and two chiral centers at the lipophilic tail region, allowing us to address the question of whether the molecular stereochemistry is related to the macroscopic phase behavior of phytantriol. In contrast to its commercial stereoisomeric mixtures, which form an isotropic micellar phase, neat (2S,3S,7R,11R)-3,7,11,15-tetramethylhexadecane-1,2,3-triol (S,S-PT) shows a smectic lamellar phase at room temperature, whereas (2R,3R,7R,11R)-3,7,11,15-tetramethylhexadecane-1,2,3-triol (R,R-PT) forms solid crystals. The lyotropic phase behavior of R,R-PT appears to be identical to that of the previously reported commercial stereoisomeric PT mixtures. In contrast, S,S-PT exhibits a different phase behavior. A lamellar crystalline phase (Lc) is formed instead of an isotropic micellar phase at a low water content, which also coexisted with other phases at low temperature. Subtle change in the shape of the diastereomers leads to variable steric interactions and subsequently affects the packing of the lipids at the molecular level, thereby influencing its self-assembling behavior. Finally, lipidic cubic phase crystallization of the membrane protein bacteriorhodopsin yielded a larger number of microcrystals with a higher average crystal length from S,S-PT than from commercial PT, suggesting faster nucleation.
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Lipidic cubic phases (LCPs) can reduce Pd2+ salts to palladium nanoparticles (PdNPs) of â¼5 nm size in their confined water channels under mild conditions. The resulting PdNP-containing LCPs were used as nanoreactor scaffolds to catalyze Suzuki-Miyaura cross-coupling reactions in the aqueous channels of the mesophase. To turn on catalysis, PdNP-containing LCPs were activated by swelling the aqueous channels of the lipidic framework, thereby enabling diffusion of the water-soluble substrates to the catalysts. The mesophases play a threefold role: they act as reducing agents for Pd2+, as limiting templates for their growth, and as support. The system was characterized and investigated by small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance. Bulk LCPs and three dispersed palladium/lipid hybrid nanoparticle types were applied in the catalysis. The latter-liposomes, hexosomes, and cubosomes-can be obtained by design through combination of lipids and additives. The Suzuki-Miyaura cross-coupling of 5-iodo-2'-deoxyuridine and phenylboronic acid was used as a model reaction to study these systems. Bulk Pd-LCPs deliver the Suzuki-Miyaura product in 24 h in conversions up to 98% at room temperature, whereas with palladium/lipid dispersions at 40 °C, 68% of the starting material was transformed to the product after 72 h.
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Functional reconstitution of membrane proteins within lipid bilayers is crucial for understanding their biological function in living cells. While this strategy has been extensively used with liposomes, reconstitution of membrane proteins in lipidic cubic mesophases presents significant challenges related to the structural complexity of the lipid bilayer, organized on saddle-like minimal surfaces. Although reconstitution of membrane proteins in lipidic cubic mesophases plays a prominent role in membrane protein crystallization, nanotechnology, controlled drug delivery, and pathology of diseased cells, little is known about the molecular mechanism of protein reconstitution and about how transport properties of the doped mesophase mirror the original molecular gating features of the reconstituted membrane proteins. In this work we design a general strategy to demonstrate correct functional reconstitution of active and selective membrane protein transporters in lipidic mesophases, exemplified by the bacterial ClC exchanger from Escherichia coli (EcClC) as a model ion transporter. We show that its correct reconstitution in the lipidic matrix can be used to generate macroscopic proton and chloride pumps capable of selectively transporting charges over the length scale of centimeters. By further exploiting the coupled chloride/proton exchange of this membrane protein and by combining parallel or antiparallel chloride and proton gradients, we show that the doped mesophase can operate as a charge separation device relying only on the reconstituted EcClC protein and an external bias potential. These results may thus also pave the way to possible applications in supercapacitors, ion batteries, and molecular pumps.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Membrana Celular/química , Canais de Cloreto/química , Proteínas de Escherichia coli/químicaRESUMO
The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air-solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air-water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur.
Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Lipídeos de Membrana/química , Nanopartículas/química , 1,2-Dipalmitoilfosfatidilcolina/químicaRESUMO
New stimuli-responsive nanomaterials, made up of host-guest lipidic cubic phases (LCPs) are presented. These biocompatible, stable, transparent and water-insoluble LCPs are composed of monoolein (MO) as a neutral host, and small amounts of one of three judiciously designed and synthesized designer lipids as guest that preserve the structure and stability of LCPs, but render them specific functionalities. Efficient pH- and light-induced binding, release and sequestration of hydrophilic dyes are demonstrated. Significantly, these processes can be performed sequentially, thereby achieving both temporal and dosage control, opening up the possibility of using such LCPs as effective carriers to be used in drug delivery applications. Specifically, because of the inherent optical transparency and molecular isotropy of LCPs they can be envisaged as light-induced drug carriers in ophthalmology. The results presented here demonstrate the potential of molecular design in creating new functional materials with predicted operating mode.
Assuntos
Lipídeos/química , Nanoestruturas/química , Sistemas de Liberação de Medicamentos , Estrutura MolecularRESUMO
Here, we present the design and assembly of a new light-responsive functional lyotropic liquid crystal system using host-guest lipidic mesophases (LMPs). Light as an external stimulus has many advantages in comparison to other stimuli: it is milder than acids or bases, and variation of intensity and duration can provide a high level of pharmacological control. The LMPs are composed of monoolein (MO) and oleic acid (OA) as host lipids and a small amount of a judiciously synthesized lipid bearing an azobenzene photoactive unit as a guest. While preserving the structure and stability of the host lipidic aggregates, the guest lipids render them specific functionalities. Single-step and sequential light-triggered release and retention of the embedded dye molecules are demonstrated, thereby achieving exquisite temporal, spatial, and dosage control of the release, opening up the possibility of using such lipidic biomaterials as effective matrices in therapy, when a continuous release of active drugs might be toxic.
Assuntos
Cristais Líquidos/química , Raios Ultravioleta , Compostos Azo/química , Cloretos/química , Glicerídeos/química , Modelos Moleculares , Conformação Molecular , Ácido Oleico/química , Compostos de Zinco/químicaRESUMO
The functionalization of cubosomes with biotin is reported here as an alternative method for the preparation of drug delivery systems capable of active targeting specific receptors that are (over)expressed by cancer cells. We describe the design, synthesis, assembly, and characterization of these novel cubosome nanoparticles by small-angle X-ray scattering (SAXS) and dynamic laser light scattering (DLS) and show their application to human adenocarcinoma cell line HeLa. These cubosomes are stabilized and functionalized with a novel, designed biotin-based block copolymer and are able to simultaneously transport paclitaxel, a potent anticancer drug, and a hydrophobic fluorescent dye in the active targeting of cancer cells. Such biotinylated cubosomes are potentially applicable in diagnosis, drug delivery, and monitoring of the therapeutic response for active targeting versus cancer cells.
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Biotina/química , Portadores de Fármacos/química , Fluoresceína/química , Corantes Fluorescentes/química , Lipídeos/química , Nanopartículas/química , Paclitaxel/química , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Células HeLa , Humanos , Cristais Líquidos/química , Modelos Moleculares , Conformação Molecular , Paclitaxel/farmacologiaRESUMO
Lyotropic liquid crystalline systems are excellent carriers for drugs due to their biocompatibility, stability in aqueous environment, and well-defined structure that allow them to host significantly larger amounts of drugs than carriers such as liposomes or gold nanoparticles. Incorporating the drug within the mesophase gel, or the cubosome/hexosome nanoparticles, decreased its toxic effects toward healthy cells, while appropriate mechanisms can stimulate the release of the drug from the carrier when it approaches the cancerous cell environment. Electrochemical methods-chronocoulometry and voltammetry at micro and normal size electrodes-are used for the first time to simultaneously determine the diffusion coefficients and effective concentrations of a toxic anticancer drug, doxorubicin, in the channels of three liquid-crystalline lipidic cubic phases. This approach was instrumental in demonstrating that the drug diffusion and kinetics of release from the mesophases depend on the aqueous channel size, which in turn is related to the identity and structure of the amphiphilic molecules used for the formation of the mesophase. Structural parameters of the cubic phases with the incorporated drug were characterized by small-angle X-ray scattering (SAXS), and molecular dynamics simulations were applied in order to describe the differences in the distribution of doxorubicin in the cubic phase matrix at acidic and neutral pH. The release of the drug from the phase was retarded at physiological pH, while at lower pH, corresponding to the cancer environment, it was accelerated, provided that suitable amphiphilic molecules were employed for the construction of the liquid crystal drug delivery system.
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Portadores de Fármacos/química , Cristais Líquidos/química , Preparações de Ação Retardada , Difusão , Doxorrubicina/química , Eletroquímica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Água/químicaRESUMO
Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small-angle X-ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high-temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP-based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C.
Assuntos
Glicerídeos/química , Proteínas de Membrana/química , Bacteriorodopsinas/química , Temperatura Baixa , Cristalização , Proteínas de Membrana/metabolismo , Transição de Fase , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Bicontinuous lipidic cubic phases (LCPs) exhibit a combination of material properties that make them highly interesting for various biomaterial applications: they are nontoxic, biodegradable, optically transparent, thermodynamically stable in excess water, and can incorporate active molecules of virtually any polarity. Here we present a molecular system comprising host lipid, water, and designed lipidic additive, which form a structured, pH-sensitive lipidic matrix for hydrophilic as well as hydrophobic drug incorporation and release. The model drug doxorubicin (Dox) was loaded into the LCP. Tunable interactions with the lipidic matrix led to the observed pH-dependent drug release from the phase. The rate of Dox release from the cubic phase at pH 7.4 was low but increased significantly at more acidic pH. A small amount of a tailored diacidic lipid (lipid 1) added to the monoolein LCP modified the release rate of the drug. Phase identity and structural parameters of pure and doped mesophases were characterized by small-angle X-ray scattering (SAXS), and release profiles from the matrix were monitored electrochemically. Analysis of the release kinetics revealed that the total amount of drug released from the LCP matrix is linearly dependent on the square root of time, implying that the release mechanism proceeds according to the Higuchi model.
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Sistemas de Liberação de Medicamentos , Lipídeos/química , Doxorrubicina/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , OxirreduçãoRESUMO
Combination of surface analytical techniques was employed to investigate the interfacial behavior of the two designed lipids-N-stearoylglycine (1) and its bulky neutral headgroup-containing derivative N-stearoylvaline ethyl ester (2)-at the air-solution interface and as transferred layers on different substrates. Formation of monolayers at the air-water interface was monitored on pure water and on aqueous solutions of different pH. Crystallization effects were visualized at pure water by recording the hystereses in the Langmuir-Blodgett (LB) isotherms and by transferring the layers onto mica, gold (111), and ITO (indium-tin oxide on glass) electrodes. Subphase pH affects the morphology and patch formation in monolayers of 1, as evidenced by BAM measurements. At pH 8.2, formation of well-ordered crystallites is observed, which upon compression elongate according to predominantly 1-D growth mechanism to form a dense layer of crystallites. This effect is not observed in monolayers of 2, whose headgroup is not protonated. The orientation of layers of 1 transferred to the solid supports is also pH dependent, and their stability can be related to formation of a hydrogen-bonded networks. AFM images of 1 exhibited platelets of multilayer phase. The IR spectra of the ITO substrates covered by 1 indicated formation of hydrogen bonds between the amide groups. The nature of the adsorption layer and its organization as a function of potential were studied in-depth by EC STM using Au(111) as the substrate. A model showing the arrangement of hydrogen bonds between adsorbed molecules is presented and related to the observed organization of the layer.
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A classical conundrum in origin-of-life studies relates to the nature of the first chemical system: was it a carrier of genetic information or a facilitator of cellular compartmentalization? Here we present a system composed of tailor-made nucleolipids and hydrated monoolein, which assemble at ambient temperatures to form host-guest lipidic cubic phase (LCP) materials that are stable in bulk water and can perform both functions. As such, they may represent a molecular model for a protocell in origin-of-life studies. Nucleolipids within the lipidic material sequester and bind selectively complementary oligonucleotide sequences from solution by virtue of base-pairing; noncomplementary sequences diffuse freely between the LCP material and the bulk aqueous environment. Sequence specific enrichment of nucleic acids within the LCP material demonstrates an effective mechanism for selection of genetic material in these cell-mimetic systems.
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Ácidos Nucleicos/química , Lipídeos/química , Modelos Moleculares , Oligonucleotídeos/química , Água/químicaRESUMO
A series of novel lipids with designed functionalities were synthesized. These lipids are based on conjugation of α-amino acids and their esters, cationic, anionic, neutral, and photochromic moieties to the lipophilic 9-cis octadecenyl chains by amide, ester, thioester, or amine bonds. Because of the plasticity of lipidic cubic phases, it is envisaged that when mixed with monooleoyl-rac-glycerol (monoolein, MO) and water at appropriate proportions, they would assemble to form bicontinuous lipidic cubic phases (LCPs) that exhibit the well-known material properties of LCPs such as phase stability, optical transparency, and chemical permeability. Moreover, due to the nature and position of the functionality at the headgroup region, we envision them to perform as functional materials by design.
Assuntos
Materiais Biocompatíveis/síntese química , Glicerídeos/química , Glicerol/química , Lipídeos/síntese química , Materiais Biocompatíveis/química , Desenho de Fármacos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Modelos BiológicosRESUMO
Colloidal lipidic particles with different space groups and geometries (mesosomes) are employed in the development of new nanosystems for the oral delivery of drugs and nutrients. Understanding of the enzymatic digestion rate of these particles is key to the development of novel formulations. In this work, the molecular structure of the lipids has been systematically tuned to examine the effect on their self-assembly and digestion rate. The kinetic and phase changes during the lipase-catalysed hydrolysis of mesosomes formed by four synthetic cyclopropanated lipids and their cis-unsaturated analogues were monitored by dynamic small angle X-ray scattering and acid/base titration. It was established that both the phase behaviour and kinetics of the hydrolysis are greatly affected by small changes in the molecular structure of the lipid as well as by the internal nanostructure of the colloidal particles.
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The aqueous channel size of lipidic cubic phases can be a limiting factor for certain applications. For this reason, additives have been used to exquisitely control their nanostructure. In this study, two families of primary phosphoesters have been designed, synthesised and utilised to determine the effect of the positioning of the guest additive at the interface of the host mesophase, and to contrast the effect of headgroup ionisation and protonation. A general methodology has been developed to produce primary phosphoesters, and a unique use of 31P NMR has been used in order to systematically investigate the influence of these additives on monoolein- and phytantriol-based bulk lipidic cubic phases and dispersed cubosomes. In general, di-phosphorylated additives exhibit a greater effect upon lipid packing than the mono- and tri-phosphorylated molecules due to their optimal positioning. In dispersion, the protonation state of the phosphate headgroups was manipulated by altering the pH, where shifts in pKa determined by 31P NMR were used as a fluorescent label-free method to identify the location and ionisation state of the phosphate additives. This study systematically evaluates the influence of the positioning of the additive, headgroup size and charge of phosphorylated lipids on the behaviour of lipidic mesophases.
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The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.
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Deferasirox/farmacologia , Quelantes de Ferro/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Permeabilidade/efeitos dos fármacosRESUMO
Water is a ubiquitous liquid with unique physicochemical properties, whose nature has shaped our planet and life as we know it. Water in restricted geometries has different properties than in bulk. Confinement can prevent low-temperature crystallization of the molecules into a hexagonal structure and thus create a state of amorphous water. To understand the survival of life at subzero temperatures, it is essential to elucidate this behaviour in the presence of nanoconfining lipidic membranes. Here we introduce a family of synthetic lipids with designed cyclopropyl modifications in the hydrophobic chains that exhibit unique liquid-crystalline behaviour at low temperature, which enables the maintenance of amorphous water down to ~10 K due to nanoconfinement. The combination of experiments and molecular dynamics simulations unveils a complex lipid-water phase diagram in which bicontinuous cubic and lamellar liquid crystalline phases that contain subzero liquid, glassy or ice water emerge as a competition between the two components, each pushing towards its thermodynamically favoured state.
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The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.
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The unique molecular architecture of lipidic cubic phases (LCPs) and their cubosome dispersions comprise a well-defined, curved bilayer that spans the entire three-dimensional (3-D) material space, encompassing a network of two periodic, curved, and nonintersecting 3-D aqueous channels. The ensuing large lipid/water interfacial area makes these biomaterials an interesting matrix for the lateral immobilization of organocatalysts to catalyze organic reactions in confined water. Herein, we report for the first time the design, synthesis, assembly, and characterization of catalytically active LCPs and cubosomes and demonstrate their applicability as self-assembled, biomimetic, and recyclable nanoreactor scaffolds. Small-angle X-ray scattering, cryo-transmission electron microscopy, and dynamic light scattering were applied for the characterization of the mesophases. These mesophases can be recycled and enable efficient catalytic activity as well as modulation of the diastereo- and enantioselectivity for the aldol reaction of several benzaldehyde derivatives and cyclohexanone in water.
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Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain.