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1.
Anal Chim Acta ; 1178: 338809, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482865

RESUMO

We present a new analytical approach for the analysis of triacylglycerol fatty acyls distribution by normal phase liquid chromatography (NPLC) coupled with APPI+-HRMS. The NPLC method used allows the separation of more than 30 classes of lipids. The energy of the APPI+ source enables the formation of low-intensity ions B fragments ([RC = O+74]+ <3%), characteristic of lipids with a glycerol esterified by one or more fatty acyls. We found the relative intensities of ions B were close to the fatty acyl distribution. To establish the proof of concept, we decided to focus on the triacylglycerols (TGs) class, the major component of plant oils. By either NPLC or FIA, the TGs class appeared as a single peak. In our experimental conditions, ions B are always present in the mass spectra of TGs and each ion B is specific to a fatty acyl group. The Orbitrap mass spectrometer featured high enough resolution and accuracy to identify ions B and distinguish them from other TG fragment ions. A further adjustment of the fatty acyls relative quantities calculation from ions B intensities was computed using weighting coefficients of ions B response. The methodology was developed and validated using plant oils characterized by a GC-FID reference method. NPLC-APPI+-HRMS method offers the advantage of analyzing the fatty acyl composition of complex lipid extracts without the need for sample preparation.


Assuntos
Pressão Atmosférica , Monoglicerídeos , Cromatografia Líquida de Alta Pressão , Lipídeos , Espectrometria de Massas , Triglicerídeos
2.
Int J Pharm ; 609: 121076, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34481886

RESUMO

A simple approach to achieve a lipoprotein (LP)-mediated drug delivery is to trigger the spontaneous drug insertion into endogenous lipoproteins in the bloodstream, by means of its chemical modification. Nanoparticles (NPs) made of the squalene-gemcitabine (SQGem) conjugate were found to have a high affinity for plasma lipoproteins while free gemcitabine did not, suggesting a key role of the lipid moiety in this event. Whether the drug conjugation to cholesterol, one of the major lipoprotein-transported lipids, could also promote an analogous interaction was a matter of question. NPs made of the cholesterol-gemcitabine conjugate (CholGem) have been herein thoroughly investigated for their blood distribution profile both in vitro and in vivo. Unexpectedly, contrarily to SQGem, no trace of the CholGem prodrug could be found in the lipoprotein fractions, nor was it interacting with albumin. The investigation of isolated NPs and NPs/LPs physical mixtures provided a further insight into the lack of interaction of CholGem NPs with LPs. Although essential for allowing the self-assembly of the prodrug into nanoparticles, the lipid moiety may not be sufficient to elicit interaction of the conjugated drug with plasma lipoproteins but the whole NP physicochemical features must be carefully considered.

3.
Biol Sex Differ ; 12(1): 52, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535195

RESUMO

BACKGROUND: The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects. METHODS: To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs. RESULTS: Cardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected. CONCLUSION: Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360941

RESUMO

Phospholipids (PLs) are amphiphilic molecules that were essential for life to become cellular. PLs have not only a key role in compartmentation as they are the main components of membrane, but they are also involved in cell signaling, cell metabolism, and even cell pathophysiology. Considered for a long time to simply be structural elements of membranes, phospholipids are increasingly being viewed as sensors of their environment and regulators of many metabolic processes. After presenting their main characteristics, we expose the increasing methods of PL detection and identification that help to understand their key role in life processes. Interest and importance of PL homeostasis is growing as pathogenic variants in genes involved in PL biosynthesis and/or remodeling are linked to human diseases. We here review diseases that involve deregulation of PL homeostasis and present a predominantly muscular phenotype.


Assuntos
Músculo Estriado/metabolismo , Fosfolipídeos/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Músculo Estriado/fisiologia , Fosfolipídeos/química
5.
Sci Adv ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523852

RESUMO

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

6.
Int J Pharm ; 584: 119391, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32376444

RESUMO

The antipsychotic drug chlorpromazine (CPZ) has potential for the treatment of acute myeloid leukemia, if central nervous system side-effects resulting from its passage through the blood-brain barrier can be prevented. A robust drug delivery system for repurposed CPZ would be drug-in-cyclodextrin-in-liposome that would redirect the drug away from the brain while avoiding premature release in the circulation. As a first step, CPZ complexation with cyclodextrin (CD) has been studied. The stoichiometry, binding constant, enthalpy, and entropy of complex formation between CPZ and a panel of CDs was investigated by isothermal titration calorimetry (ITC). All the tested CDs were able to include CPZ, in the form of 1:1, 1:2 or a mixture of 1:1 and 1:2 complexes. In particular, a substituted γ-CD, sugammadex (the octasodium salt of octakis(6-deoxy-6-S-(2-carboxyethyl)-6-thio)cyclomaltooctaose), formed exclusively 1:2 complexes with an extremely high association constant of 6.37 × 109 M-2. Complexes were further characterized by heat capacity changes, one- and two-dimensional (ROESY) nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Finally, protection of CPZ against photodegradation by CDs was assessed. This was accelerated rather than reduced by complexation with CD. Altogether these results provide a molecular basis for the use of CD in delayed release formulations for CPZ.


Assuntos
Química Farmacêutica/métodos , Clorpromazina/administração & dosagem , Ciclodextrinas/química , Lipossomos/química , Clorpromazina/química , Preparações de Ação Retardada , Portadores de Fármacos/química , Reposicionamento de Medicamentos , Estabilidade de Medicamentos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Sugammadex/química , Termodinâmica , beta-Ciclodextrinas/química , gama-Ciclodextrinas/química
7.
Anal Bioanal Chem ; 412(3): 777-793, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31858168

RESUMO

Stratum corneum lipids are responsible for the skin's barrier function. They are the final product of epidermis lipid biosynthesis. During this process, lipids evolve from simple to complex structures in three main levels respectively (stratum basal level, stratum granulosum level, and stratum corneum level). Our aim was to simultaneously analyze and characterize the structure of total epidermis lipids. A powerful analytical method (normal-phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MSn)) was developed in order to separate, in a single run, lipid classes with a wide polarity range. Chromatographic conditions were particularly designed to analyze lipids of intermediate polarity such as ceramides. Rich information was obtained about the molecular structure of keratinocyte differentiation biomarkers such as ceramides, glucosylceramides, and sphingomyelins and the microstructures of reconstructed human epidermis lipids using HR-MSn. A new subclass of ceramides, 1-O-Acyl Omega-linoleoyloxy ceramides [1-O-E (EO) Cer] has been highlighted. This class is double esterified on the 1-O-position of sphingoid base with long to very long chain acyl residues (1-O-E) and on the position of ω-hydroxyl group of fatty acid with the linolenic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. In addition, we detected a new epidermis sphingomyelins. Our lipidomic approach offers a direct access to epidermis biomarkers.


Assuntos
Ceramidas/análise , Cromatografia Líquida/métodos , Epiderme/química , Lipídeos/análise , Espectrometria de Massas/métodos , Humanos
8.
Cell Rep ; 29(12): 3974-3982.e4, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851927

RESUMO

The essentiality of fatty acid synthesis (FASII) products in the human pathogen Staphylococcus aureus is the underlying rationale for FASII-targeted antimicrobial drug design. Reports of anti-FASII efficacy in animals support this choice. However, restricted test conditions used previously led us to investigate this postulate in a broader, host-relevant context. We report that S. aureus rapidly adapts to FASII antibiotics without FASII mutations when exposed to host environments. FASII antibiotic administration upon signs of infection, rather than just after inoculation as commonly practiced, fails to eliminate S. aureus in a septicemia model. In vitro, serum lowers S. aureus membrane stress, leading to a greater retention of the substrates required for environmental fatty acid (eFA) utilization: eFAs and the acyl carrier protein. In this condition, eFA occupies both phospholipid positions, regardless of anti-FASII selection. Our results identify S. aureus membrane plasticity in host environments as a main limitation for using FASII antibiotics in monotherapeutic treatments.


Assuntos
Adaptação Fisiológica , Antibacterianos/farmacologia , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Sepse/patologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Farmacorresistência Bacteriana , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Sepse/tratamento farmacológico , Sepse/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
9.
Microb Cell Fact ; 18(1): 131, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400768

RESUMO

BACKGROUND: The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored. RESULTS: In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG). CONCLUSIONS: In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Lipídeos/química , Proteínas de Membrana/biossíntese , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Engenharia Metabólica , Canais de Translocação SEC/química , Canais de Translocação SEC/genética
10.
Arch Dermatol Res ; 310(7): 579-589, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29995261

RESUMO

Few studies have investigated the influence of increased amounts of dietary linoleic acid on the epidermal lipid biochemistry and TEWL in healthy subject. The influence of dietary linoleic acid on canine stratum corneum (SC) lipids was studied by feeding two groups of five dogs differential amounts of linoleic acid (LA) for three months. SC was harvested by tape stripping and lipids were analyzed by thin-layer chromatography and mass spectrometry. The dogs that were fed the higher concentration of LA showed high increases in the contents of both linoleic acid and free ceramides in the SC, whereas the protein-bound ceramide content was unchanged. Acylacids that represent the esterified form of linoleic acid in omega hydroxy very long chain fatty acids (ω-OH VLCFA) accounted for most of the elevation of LA, whereas the concentration of the free form was not significantly changed. Corroborating the absence of change in the protein-bound ceramides content of healthy dogs SC, TEWL was nearly unaffected by the linoleic acid-enriched diet.


Assuntos
Epiderme/metabolismo , Ácido Linoleico/metabolismo , Animais , Ceramidas/metabolismo , Dietoterapia , Cães , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos , Perda Insensível de Água
11.
Sci Total Environ ; 624: 1082-1094, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29625523

RESUMO

This study employed a UV-A/visible/TiO2 system to investigate the degradation of pemetrexed, an antifolate agent used in chemotherapy. The laboratory-scale method employed a photostability chamber that could be used to study multiple samples. Reversed-phase HPLC coupled with high-resolution ESI-LTQ-Orbitrap mass spectrometry was used to determine the transformation products (TPs) of PEME. Based on the identified TPs and existing chemical knowledge, the mechanism of degradation of the target compound was proposed. Concentrations were monitored as a function of time, and the degradation kinetics were compared. The structures of seven TPs, four of which have not been described to date, were proposed. Most of the TPs stemmed from OH radical additions to the dihydropyrrole moiety and oxidative decarboxylation of the glutamate residue. Based on the elucidated structures, a computational toxicity assessment was performed, showing that the TPs with higher log D values than the parent compound are more toxic than the PEME itself. To support these findings, the toxicities of irradiated samples on Vibrio fischeri were monitored over time. The experimental results corresponded well with the results of previous computational studies.

12.
J Pharm Biomed Anal ; 152: 31-38, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29414016

RESUMO

In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and l-glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC-MS/MS quantification of l-glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core-shell ammonium-sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25 mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of l-glutamic acid, glutamine and GABA, as well as the internal standards [d6]-GABA and [d5]-glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63 ng/ml for GABA, 1.25 ng/ml for l-glutamic acid and 3.15 ng/ml for glutamine, and the intra-day and inter-day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium-evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.


Assuntos
Lobo Frontal/química , Ácido Glutâmico/química , Glutamina/química , Ácido gama-Aminobutírico/química , Acetonitrilas/química , Compostos de Amônio/química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise/métodos , Neurotransmissores/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácidos Sulfônicos/química , Espectrometria de Massas em Tandem/métodos
13.
J Chromatogr A ; 1514: 54-71, 2017 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-28774713

RESUMO

One important challenge in lipid class analysis is to develop a method suitable or, at least adaptable, for a vast diversity of samples. In the current study, an improved normal-phase liquid chromatography (NPLC) method allowed analyzing the lipid classes present in mammalian, vegetable as well as microorganism (yeast and bacteria) lipid samples. The method effectively separated 30 lipid classes or subclasses with a special focus on medium polarity lipids. The separation was carried out with bare silica stationary phase and was coupled to evaporative light scattering detection (ELSD), charged aerosol detection (Corona-CAD®) and mass spectrometry. Solutions are provided to circumvent technical issues (such as pumping solvents of low viscosity, solvent purity, rinsing step). The influence of mobile phase composition and addition of ionic modifiers on the chromatographic behavior of particular lipid classes is documented. A comparison between ELSD and Corona-CAD® confirmed the interest of this later detector for samples with a wide range of concentration of different lipids. Three common atmospheric pressure ionization interfaces were used for coupling the NPLC separation to a LTQ Velos Pro® mass spectrometer. The comparison of the chromatographic profiles showed that atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are both suitable to detect the different lipid classes whereas APPI allows a better sensitivity for lipids at low-concentration.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/análise , Espectrometria de Massas por Ionização por Electrospray , Aerossóis/química , Animais , Pressão Atmosférica , Encéfalo/metabolismo , Bovinos , Galinhas , Gema de Ovo/química , Gema de Ovo/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Íons/química , Lipídeos/química , Fígado/química , Fígado/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Solventes/química , Soja/química , Soja/metabolismo
14.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(10 Pt A): 1079-1091, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28739279

RESUMO

A diet containing a high n-3/n-6 polyunsaturated fatty acids (PUFA) ratio has cardioprotective properties. PUFAs incorporation into membranes influences the function of membrane proteins. We investigated the impact of the membrane incorporation of PUFAs, especially eicosapentaenoic acid (EPA) (C20:5 n-3), on the anti-atherogenic cholesterol efflux pathways. We used cholesteryl esters (CE)-loaded human monocyte-derived macrophages (HMDM) to mimic foam cells exposed to the FAs for a long period of time to ensure their incorporation into cellular membranes. Phospholipid fraction of EPA cells exhibited high levels of EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which was associated with a decreased level of arachidonic acid (AA) (C20:4 n-6). EPA 70µM reduced ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 30% without any alteration in ABCA1 expression. The other tested PUFAs, DPA, docosahexaenoic acid (DHA) (C22:6 n-3), and AA, were also able to reduce ABCA1 functionality while the monounsaturated oleic FA slightly decreased efflux and the saturated palmitic FA had no impact. Moreover, EPA also reduced cholesterol efflux to HDL mediated by the Cla-1 and ABCG1 pathways. EPA incorporation did not hinder efflux in free cholesterol-loaded HMDM and did not promote esterification of cholesterol. Conversely, EPA reduced the neutral hydrolysis of cytoplasmic CE by 24%. The reduced CE hydrolysis was likely attributed to the increase in cellular TG contents and/or the decrease in apo E secretion after EPA treatment. In conclusion, EPA membrane incorporation reduces cholesterol efflux in human foam cells by reducing the cholesteryl ester mobilization from lipid droplets.


Assuntos
Membrana Celular/metabolismo , Ésteres do Colesterol/metabolismo , Ácido Eicosapentaenoico , Gotículas Lipídicas/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/biossíntese , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Ácido Eicosapentaenoico/farmacocinética , Ácido Eicosapentaenoico/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas HDL/metabolismo , Masculino , Receptores Depuradores Classe B/biossíntese
15.
Biochim Biophys Acta Biomembr ; 1859(6): 1124-1132, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28284722

RESUMO

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.


Assuntos
Proteínas de Bactérias/metabolismo , Cardiolipinas/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/deficiência , Mitocôndrias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Proteínas de Bactérias/genética , ATPases Bacterianas Próton-Translocadoras/genética , ATPases Bacterianas Próton-Translocadoras/metabolismo , Retículo Endoplasmático/ultraestrutura , Escherichia coli/ultraestrutura , Corantes Fluorescentes/química , Deleção de Genes , Expressão Gênica , Isoenzimas/deficiência , Isoenzimas/genética , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Imagem com Lapso de Tempo , Transferases (Outros Grupos de Fosfato Substituídos)/genética
16.
Pharm Res ; 34(5): 1067-1082, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28168390

RESUMO

PURPOSE: The aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity. METHODS: Micro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic® F127 20 wt% hydrogel. RESULTS: The formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC50 and the MIC90 decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI = 0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC. CONCLUSION: These results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Plaquetas/química , Candida albicans/efeitos dos fármacos , Quitosana/farmacologia , Ácido Desoxicólico/farmacologia , Anfotericina B/química , Animais , Antifúngicos/química , Candidíase/tratamento farmacológico , Química Farmacêutica/métodos , Quitosana/química , Ácido Desoxicólico/química , Modelos Animais de Doenças , Combinação de Medicamentos , Feminino , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Membrana Mucosa/efeitos dos fármacos , Nanopartículas/química , Poloxâmero/química , Suínos , alfa-Ciclodextrinas/química
17.
Biochim Biophys Acta ; 1858(11): 2725-2736, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27457703

RESUMO

Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts.


Assuntos
Membrana Celular/química , Colesterol/química , Células Epiteliais/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , Cromatografia Líquida , Células Epiteliais/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Membranas Artificiais , Mimetismo Molecular , Fosfolipídeos/classificação , Fosfolipídeos/metabolismo , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/metabolismo , Espectrometria de Massas em Tandem
18.
Biol Sex Differ ; 6: 20, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26478810

RESUMO

BACKGROUND: Imbalance in lipid metabolism and membrane lipid homeostasis has been observed in numerous diseases including heart failure and cardiotoxicity. Growing evidence links phospholipid alterations especially cardiolipins (CLs) to defects in mitochondrial function and energy metabolism in heart failure. We have shown recently that doxorubicin cardiotoxicity is more severe in male than female Wistar rats. We aimed to study whether this sex specificity is linked to differences in cardiac phospholipid profiles. RESULTS: Adult male and female rats were injected 2 mg/kg doxorubicin weekly for 7 weeks. Cardiac phospholipid molecular species were determined by liquid chromatography coupled with mass spectrometry fragmentation (LC)/MS(n). Sex difference in phosphatidylethanolamine and phosphatidylcholine species containing docosahexaenoic and docosapentaenoic acyl chains was observed, females having more than males. In both sexes, doxorubicin induced an important loss of the main CL(18:2)4, while the level of monolysocardiolipin MLCL(18:2)3 remained stable. However, a severe remodelling appeared in treated rats with the longest CL acyl chains in doxorubicin-treated females, which might compensate for the loss of tetra-linoleoyl CL. The level of oxidized cardiolipin was not particularly increased after doxorubicin treatment. Finally, expression of genes involved in the biosynthesis of fatty acid appeared to be decreased in doxorubicin-treated males. CONCLUSIONS: These results emphasize for the first time the cardiac remodelling in the phospholipid classes after doxorubicin treatment. These observations suggest that doxorubicin has a sex-specific impact on the heart phospholipidome especially on cardiolipin, an essential mitochondrial lipid. Further studies are needed to better understand the roles of lipids in the anthracycline cardiotoxicity and sex differences, but phospholipid cardioprotection seems a valuable new additive therapeutic strategy for anthracycline cardiotoxicity.

19.
Circ Heart Fail ; 8(1): 98-108, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25420486

RESUMO

BACKGROUND: Cardiovascular diseases are the major cause of mortality among both men and women with a lower incidence in women before menopause. The clinical use of doxorubicin, widely used as an antineoplastic agent, is markedly hampered by severe cardiotoxicity. Even if there is a significant sex difference in incidence of cardiovascular disease at the adult stage, it is not known whether a difference in doxorubicin-related cardiotoxicity between men and women also exists. The objective of this work was to explore the cardiac side effects of doxorubicin in adult rats and decipher whether signaling pathways involved in cardiac toxicity differ between sexes. METHODS AND RESULTS: After 7 weeks of doxorubicin (2 mg/kg per week), males developed major signs of cardiomyopathy with cardiac atrophy, reduced left ventricular ejection fraction and 50% mortality. In contrast, no female died and their left ventricular ejection fraction was only moderately affected. Surprisingly, neither global oxidation levels nor the antioxidant response nor the apoptosis signaling pathways were altered by doxorubicin. However, the level of total adenosine monophosphate-activated protein kinase was severely decreased only in males. Moreover, markers of mitochondrial biogenesis and cardiolipin content were strongly reduced only in males. To analyze the onset of the pathology, maximal oxygen consumption rate of left ventricular permeabilized fibers after 4 weeks of treatment was reduced only in doxorubicin-treated males. CONCLUSIONS: Altogether, these results clearly evidence sex differences in doxorubicin toxicity. Cardiac mitochondrial dysfunction and adenosine monophosphate-activated protein kinase seem as critical sites of sex differences in cardiotoxicity as evidenced by significant statistical interactions between sex and treatment effects.


Assuntos
Doxorrubicina/toxicidade , Metabolismo Energético/efeitos dos fármacos , Insuficiência Cardíaca/induzido quimicamente , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Índice de Massa Corporal , Cardiotoxicidade , Modelos Animais de Doenças , Feminino , Seguimentos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Ratos , Ratos Wistar , Fatores Sexuais
20.
J Pharm Biomed Anal ; 88: 542-51, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24211706

RESUMO

The quality assessment of African traditional herbal medicinal products is a difficult challenge since they are complex mixtures of several herbal drug or herbal drug preparations. The plant source is also often unknown and/or highly variable. Plant metabolites chromatographic profiling is therefore an important tool for quality control of such herbal products. The objective of this work is to propose a protocol for sample preparation and gas chromatographic profiling of non-polar metabolites for quality control of African traditional herbal medicinal products. The methodology is based on the chemometric assessment of chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber by high temperature gas chromatography coupled to mass spectrometry, performed on extracts obtained in refluxed dichloromethane, after removal of chlorophyll pigments. The method using high temperature gas chromatography after dichloromethane extraction allows detection of most non-polar bioactive and non-bioactive metabolites already identified in leaves of both species. Chemometric data analysis using Principal Component Analysis and Partial Least Squares after Orthogonal Signal Correction applied to chromatographic profiles of leaves of Combretum micranthum and Mitracarpus scaber showed slight batch to batch differences, and allowed clear differentiation of the two herbal extracts.


Assuntos
Medicina Tradicional Africana/métodos , Preparações de Plantas/análise , Plantas Medicinais/química , África , Artefatos , Técnicas de Química Analítica , Clorofila/análise , Clorofila/química , Combretum/química , Cromatografia Gasosa-Espectrometria de Massas , Análise dos Mínimos Quadrados , Cloreto de Metileno/química , Folhas de Planta/química , Análise de Componente Principal , Controle de Qualidade , Temperatura
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