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1.
Biochim Biophys Acta Biomembr ; 1864(1): 183729, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34506796

RESUMO

Fluorescence spectroscopy is used to characterize the partition of three second-generation D,L-α-cyclic peptides to two lipid model membranes. The peptides have proven antimicrobial activity, particularly against Gram positive bacteria, and the model membranes are formed of either with 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) or its mixture with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), at a molar ratio of (1:1). The peptide's intrinsic fluorescence was used in the Steady State and/or Time Resolved Fluorescence Spectroscopy experiments, showing that the peptides bind to the membranes, and the extent of their partition is thereof quantified. The peptide-induced membrane leakage was followed using an encapsulated fluorescent dye. Overall, the partition is mainly driven by electrostatics, but also involves hydrophobic interactions. The introduction of a hydrocarbon tail in one of the residues of the parent peptide, CPR, adjacent to the tryptophan (Trp) residue, significantly improves the partition of the modified peptides, CPRT10 and CPRT14, to both membrane systems. Further, we show that the length of the tail is the main distinguishing factor for the extension of the partition process. The parent peptide induces very limited leakage, at odds with the peptides with tail, that promote fast leakage, increasing in most cases with peptide concentration, and being almost complete for the highest peptide concentration and negatively charged membranes. Overall, the results help the unravelling of the antimicrobial action of these peptides and are well in line with their proven high antimicrobial activity.


Assuntos
Antibacterianos/química , Lipídeos de Membrana/química , Peptídeos Cíclicos/química , Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Membranas/química , Peptídeos Cíclicos/farmacologia , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Espectrometria de Fluorescência
2.
J Agric Food Chem ; 69(45): 13628-13636, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34739242

RESUMO

Cinnamaldehyde is a natural antimicrobial food preservative. Previous studies have suggested that cinnamaldehyde interacts with the cell membrane, but the molecular targets of cinnamaldehyde action on foodborne pathogens are still unclear. In this study, the structural changes of Staphylococcus aureus and Escherichia coli cells were observed after cinnamaldehyde treatment. Then, quantitative real-time polymerase chain reaction (PCR) and parallel reaction monitoring were used for determining the effects of cinnamaldehyde treatment of these bacteria on the expression of genes and proteins associated with glycerophospholipid biosynthesis. Changes in fatty acids (raw materials for the biosynthesis of glycerophospholipids) and glycerophospholipids in S. aureus and E. coli after cinnamaldehyde treatment were analyzed to confirm the results of gene and protein expression experiments. Cinnamaldehyde regulated the glycerophospholipid biosynthesis pathways of these foodborne pathogens, mainly targeting phosphatidylglycerol and phosphatidylethanolamine, which resulted in the disruption of cell membrane integrity.


Assuntos
Anti-Infecciosos , Staphylococcus aureus , Acroleína/análogos & derivados , Acroleína/farmacologia , Antibacterianos/farmacologia , Escherichia coli/genética , Fosfatidiletanolaminas , Fosfatidilgliceróis
3.
FEBS Lett ; 595(21): 2701-2714, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34633077

RESUMO

Several antimicrobial peptides, including magainin and the human cathelicidin LL-37, act by forming pores in bacterial membranes. Bacteria such as Staphylococcus aureus modify their membrane's cardiolipin composition to resist such types of perturbations that compromise their membrane stability. Here, we used molecular dynamic simulations to quantify the role of cardiolipin on the formation of pores in simple bacterial-like membrane models composed of phosphatidylglycerol and cardiolipin mixtures. Cardiolipin modified the structure and ordering of the lipid bilayer, making it less susceptible to mechanical changes. Accordingly, the free-energy barrier for the formation of a transmembrane pore and its kinetic instability augmented by increasing the cardiolipin concentration. This is attributed to the unfavorable positioning of cardiolipin near the formed pore, due to its small polar head and bulky hydrophobic body. Overall, our study demonstrates how cardiolipin prevents membrane-pore formation and this constitutes a plausible mechanism used by bacteria to act against stress perturbations and, thereby, gain resistance to antimicrobial agents.


Assuntos
Membrana Celular , Fosfatidilgliceróis , Cardiolipinas , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Staphylococcus aureus
4.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638772

RESUMO

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Fosfatidilgliceróis/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/metabolismo
5.
Nat Commun ; 12(1): 6193, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702812

RESUMO

Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.


Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Citotoxinas/metabolismo , Citotoxinas/toxicidade , Humanos , Leucocidinas/metabolismo , Leucocidinas/toxicidade , Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Lisina/genética , Lisina/metabolismo , Camundongos , Fagócitos/efeitos dos fármacos , Fosfatidilgliceróis/genética , Fosfatidilgliceróis/metabolismo , Transporte Proteico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Ácidos Teicoicos/genética , Ácidos Teicoicos/metabolismo , Fatores de Virulência/toxicidade
7.
Int J Hyperthermia ; 38(1): 1415-1424, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34581259

RESUMO

PURPOSE: Recommended treatments for muscle-invasive bladder cancer (MIBC) come with considerable morbidity. Hyperthermia (HT) triggered drug release from phosphatidylglycerol-based thermosensitive liposomes (DPPG2-TSL) might prevent surgical bladder removal and toxicity from systemic chemotherapy. We aimed to assess the efficacy of DPPG2-TSL with HT in a syngeneic orthotopic rat urothelial carcinoma model. METHODS: A total of 191 female Fischer F344 rats were used. Bladder tumors were initiated by inoculation of AY-27 cells and tumor-bearing rats were selected with cystoscopy and semi-randomized over treatment groups. On days 5 and 8, animals were treated with DOX in different treatment modalities: intravenous (iv) DPPG2-TSL-DOX with HT, iv free DOX without HT, intravesical DOX without HT, intravesical DOX with HT or no treatment (control group), respectively. Animals were euthanized on day 14 and complete tumor response was assessed by histopathological evaluation. RESULTS: Iv DPPG2-TSL-DOX + HT resulted in a favorable rate of animals with complete tumor response (70%), compared to iv free DOX (18%, p = .02), no treatment (0%, p = .001), and intravesical DOX with (43%, p = .35) or without HT (50%, p = .41). All rats receiving intravesical DOX with HT and 24% of rats treated with DPPG2-TSL-DOX containing the same DOX dose with HT had to be euthanized before day 14 because of substantial bodyweight loss, which was associated with dilated ureters urine retention in a few rats. CONCLUSION: Treatment with DPPG2-TSL-DOX combined with intravesical HT outperformed systemic and intravesical DOX in vivo. There might be a role for DPPG2-TSL encapsulating chemotherapeutics in the treatment of MIBC in the future.


Assuntos
Carcinoma de Células de Transição , Hipertermia Induzida , Neoplasias da Bexiga Urinária , Animais , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Lipossomos , Músculos , Fosfatidilgliceróis , Ratos , Ratos Endogâmicos F344 , Neoplasias da Bexiga Urinária/tratamento farmacológico
8.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360998

RESUMO

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Oligopeptídeos/farmacologia , Antifúngicos/química , Candida albicans/fisiologia , Cardiolipinas/metabolismo , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Oligopeptídeos/química , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Ácidos Teicoicos/metabolismo , Triptofano/química
9.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443547

RESUMO

Phosphatidylglycerols represent a large share of the lipids in the plasmamembrane of procaryotes. Therefore, this study investigates the role of charged lipids in the plasma membrane with respect to the interaction of the antiviral saponin glycyrrhizin with such membranes. Glycyrrhizin is a natural triterpenic-based surfactant found in licorice. Vesicles made of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1'-glycerol) (DOPG)/glycyrrhizin are characterized by small-angle scattering with neutrons and X-rays (SANS and SAXS). Small-angle scattering data are first evaluated by the model-independent modified Kratky-Porod method and afterwards fitted by a model describing the shape of small unilamellar vesicles (SUV) with an internal head-tail contrast. Complete miscibility of DOPG and glycyrrhizin was revealed even at a ratio of lipid:saponin of 1:1. Additional information about the chain-chain correlation distance of the lipid/saponin mixtures in the SUV structures is obtained from wide-angle X-ray scattering (WAXS).


Assuntos
Microscopia Crioeletrônica , Ácido Glicirrízico/química , Fosfatidilgliceróis/química , Espalhamento a Baixo Ângulo , Difração de Nêutrons , Difração de Raios X
10.
Colloids Surf B Biointerfaces ; 208: 112054, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34454365

RESUMO

Isoniazid (INH) is one of the primary drugs used in tuberculosis treatment and its encapsulation in liposomal vesicles can both improve its therapeutic index and minimize toxicity. Here we consider mixtures of hydrogenated soy phosphatidylcholine-phosphatidylglycerol (HSPC-DPPG) to get novel biocompatible liposomes for INH delivery. We determined INH encapsulation efficiency by coupling for the first time UV and Laser Transmission Spectroscopy and we showed that HSPC-DPPG liposomes can load more INH than expected from simple geometrical arguments, thus suggesting the presence of drug-lipid association. To focus on this aspect, which has never been explored in liposomal formulations, we employed several complementary techniques, such as dynamic and static light scattering, calorimetry and surface pressure measurements on lipid monolayers. We find that INH-lipid interaction increases the entrapment capability of liposomes due to INH adsorption. Moreover, the preferential INH-HSPC dipole-dipole interaction promotes the modification of lipid ordering, favoring the formation of HSPC-richer domains in excess of DPPG. Our findings highlight how investigating the fundamental aspects of drug-lipid interactions is of paramount importance for the optimal design of liposomal nanocarriers.


Assuntos
Isoniazida , Lipossomos , Antituberculosos , Composição de Medicamentos , Fosfatidilgliceróis
11.
Animal ; 15(8): 100280, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34252722

RESUMO

Milk fats are vital to neonate survival and development, but vary highly by diet, maternal metabolic state and stage of lactation. To gain a better understanding of changes in lipid composition of sow milk across lactation, milk was collected from nine multiparous sows on days 0, 3, 7, and 14, relative to birth and lipids were profiled using multiple reaction monitoring (MRM) profiling. Percent fat was determined by creamatocrit, and found to be different (P < 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and day 14 (12.13 ± 2.45%). Fat was extracted from milk (n = 6/day) using the Bligh-Dyer method and profiled using tandem mass spectrometry MRM to determine the abundance of lipids defined by class and fatty acyl residue composition. Lipid species relative concentration was calculated from internal standards, and data analysis was performed using Metaboanalyst 4.0. Concentration of phosphatidyl-choline, -serine, -ethanolamine, -inositol, cholesteryl ester and sphingomyelin did not vary across lactation days, nor did the distribution of associated fatty acyl residues. The total abundance of triacylglycerides (TGs) and phosphatidylglycerols (PGs) increased (P < 0.05) from colostrum (day 0) to transitional (days 3 and 7) and mature milk (day 14). As lactation days increased from day 0 to day 14, the number of carbons and unsaturation within fatty acyl residues decreased (P < 0.05) in both TGs and PGs. The proportion of TGs and PGs increased (P < 0.05) relative to other lipid classes. Changes in composition of milk triglycerides and phosphatidylglycerols likely reflect the metabolic activity of the mammary gland and developmental needs of neonates.


Assuntos
Leite , Fosfolipídeos , Animais , Membrana Celular , Colostro , Dieta , Feminino , Lactação , Lipidômica , Fosfatidilgliceróis , Gravidez , Suínos , Triglicerídeos
12.
Biochim Biophys Acta Biomembr ; 1863(11): 183698, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34283999

RESUMO

Hexadecylphosphocholine (HePC, Miltefosine) is a drug from the class of alkylphosphocholines with an antineoplastic and antiprotozoal activity. We previously reported that HePC uptake from thermosensitive liposomes (TSL) containing 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) into cancer cells is accelerated at mild hyperthermia (HT) resulting in increased cytotoxicity. In this study, we compared HePC release of different TSL formulations in serum. HePC showed rapid but incomplete release below the transition temperature (Tm) of investigated TSL formulations in serum. Short heating (5 min) to 42 °C increased HePC release from DPPG2-TSL (Tm = 41 °C) by a factor of two in comparison to body temperature (37 °C). Bovine serum albumin (BSA) induced HePC release from DPPG2-TSL comparable to serum. Furthermore, multilamellar vesicles (MLV) were capable to extract HePC from DPPG2-TSL in a concentration- and temperature-dependent manner. Repetitive exposure of DPPG2-TSL to MLV at 37 °C led to a fast initial release of HePC which slowed down after subsequent extraction cycles finally reaching approx. 50% HePC release. A pharmacokinetic study in rats revealed a biphasic pattern with an immediate clearance of approx. 50% HePC whereas the remaining 50% HePC showed a prolonged circulation time. We speculate that HePC located in the external leaflet of DPPG2-TSL is rapidly released upon contact with suitable biological acceptors. As demonstrated by MLV transfer experiments, asymmetric incorporation of HePC into the internal leaflet of DPPG2-TSL might improve HePC retention in presence of complex biological media and still give rise to HT-induced HePC release.


Assuntos
Antineoplásicos/farmacocinética , Lipossomos , Fosfatidilgliceróis/química , Fosforilcolina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Varredura Diferencial de Calorimetria , Cromatografia Líquida/métodos , Hemólise/efeitos dos fármacos , Humanos , Masculino , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologia , Ratos , Espectrometria de Massas em Tandem/métodos , Temperatura
13.
Biophys J ; 120(17): 3776-3786, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280369

RESUMO

Identification, visualization, and quantitation of cardiolipin (CL) in biological membranes is of great interest because of the important structural and physiological roles of this lipid. Selective fluorescent detection of CL using noncovalently bound fluorophore 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenylethene (TTAPE-Me) has been recently proposed. However, this dye was only tested on wild-type mitochondria or liposomes containing negligible amounts of other anionic lipids, such as phosphatidylglycerol (PG) and phosphatidylserine (PS). No clear preference of TTAPE-Me for binding to CL compared to PG and PS was found in our experiments on artificial liposomes, Escherichia coli inside-out vesicles, or Saccharomyces cerevisiae mitochondria in vitro or in situ, respectively. The shapes of the emission spectra for these anionic phospholipids were also found to be indistinguishable. Thus, TTAPE-Me is not suitable for detection, visualization, and localization of CL in the presence of other anionic lipids present in substantial physiological amounts. Our experiments and complementary molecular dynamics simulations suggest that fluorescence intensity of TTAPE-Me is regulated by dynamic equilibrium between emitting dye aggregates, stabilized by unspecific but thermodynamically favorable electrostatic interactions with anionic lipids, and nonemitting dye monomers. These results should be taken into consideration when interpreting past and future results of CL detection and localization studies with this probe in vitro and in vivo. Provided methodology emphasizes minimal experimental requirements, which should be considered as a guideline during the development of novel lipid-specific probes.


Assuntos
Cardiolipinas , Fosfolipídeos , Ânions , Lipossomos , Fosfatidilgliceróis
14.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064353

RESUMO

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Diglicerídeos/metabolismo , Glicolipídeos/metabolismo , Fosfatidilgliceróis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Galactolipídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/citologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Mutação , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Sementes/citologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
15.
Int J Nanomedicine ; 16: 4045-4061, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34163158

RESUMO

Purpose: Previous studies demonstrated the possibility of targeting tumor-angiogenic endothelial cells with positively charged nanocarriers, such as cationic liposomes. We investigated the active targeting potential of positively charged nanoparticles in combination with the heat-induced drug release function of thermosensitive liposomes (TSL). This novel dual-targeted approach via cationic TSL (CTSL) was thoroughly explored using either a novel synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) or a conventional polyethylene glycol (PEG) surface modification. Anionic particles containing either DPPG2 or PEG were also included in the study to highlight difference in tumor enrichment driven by surface charge. With this study, we aim to provide a deep insight into the main differences between DPPG2- and PEG-functionalized liposomes, focusing on the delivery of a well-known cytotoxic drug (doxorubicin; DOX) in combination with local hyperthermia (HT, 41-43°C). Materials and Methods: DPPG2- and PEG-based cationic TSLs (PG2-CTSL/PEG-CTSL) were thoroughly analyzed for size, surface charge, and heat-triggered DOX release. Cancer cell targeting and DOX delivery was evaluated by FACS, fluorescence imaging, and HPLC. In vivo particle behavior was analyzed by assessing DOX biodistribution with local HT application in tumor-bearing animals. Results: The absence of PEG in PG2-CTSL promoted more efficient liposome-cell interactions, resulting in a higher DOX delivery and cancer cell toxicity compared with PEG-CTSL. By exploiting the dual-targeting function of CTSLs, we were able to selectively trigger DOX release in the intracellular compartment by HT. When tested in vivo, local HT promoted an increase in intratumoral DOX levels for all (C)TSLs tested, with DOX enrichment factors ranging from 3 to 14-fold depending on the type of formulation. Conclusion: Cationic particles showed lower hemocompatibility than their anionic counterparts, which was partially mitigated when PEG was grafted on the liposome surface. DPPG2-based anionic TSL showed optimal local drug delivery compared to all other formulations tested, demonstrating the potential advantages of using DPPG2 lipid in designing liposomes for tumor-targeted applications.


Assuntos
Comunicação Celular , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Fosfatidilgliceróis/química , Polietilenoglicóis/química , Temperatura , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Concentração Inibidora 50 , Espaço Intracelular/metabolismo , Lipossomos , Polietilenoglicóis/administração & dosagem , Ratos , Propriedades de Superfície , Distribuição Tecidual/efeitos dos fármacos
16.
J Mol Biol ; 433(15): 167057, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34033821

RESUMO

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.


Assuntos
Cardiolipinas/metabolismo , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/metabolismo , Fosfatidilgliceróis/metabolismo , Citocromos c/metabolismo , Regulação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
17.
Biochim Biophys Acta Biomembr ; 1863(9): 183649, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33991503

RESUMO

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.


Assuntos
Corantes Fluorescentes/química , Nanopartículas/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Compostos de Piridínio/química , Lipossomos/química , Simulação de Dinâmica Molecular , Estrutura Molecular , Espectrometria de Fluorescência
18.
PLoS One ; 16(5): e0251690, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989363

RESUMO

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (kp) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σc) under different Π. The σc dependent kp values were fitted with a theoretical equation, and the corresponding membrane tension (σoseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σoseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σc and Π induced pore formation in the lipid membranes of GUVs.


Assuntos
Eletroporação , Pressão Osmótica , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Lipossomas Unilamelares/química
19.
Nat Commun ; 12(1): 2927, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006869

RESUMO

As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.


Assuntos
Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Lisina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Fosfatidilgliceróis/química , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Rhizobium tropici/genética , Rhizobium tropici/metabolismo
20.
J Liposome Res ; 31(4): 409-419, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33944651

RESUMO

Sickle cell disease (SCD) is a mortal erythrocyte-based disease which is hard to treat effectively. Development of a treatment method that can prevent deoxygenation of erythrocytes or reduce the oxidative stress of sickle erythrocytes is one of the important issues towards SCD. Among a wide variety of potential drug carriers, liposomes are advantageous and preferable with their easy preparation and biocompatibility. In this study, L-Glutamine (Gln) loaded liposomes were prepared with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG). Liposomes were characterized via zeta potential, size measurements, differential scanning calorimetry, Fourier Transform Infra-red Spectroscopy and they were visualized via transmission electron microscopy and scanning electron microscopy. Effect of the encapsulated amount of Gln was investigated by encapsulating Gln at three different concentrations (i.e0.20 mM, 40 mM and 60 mM). Drug encapsulation and release studies were implemented with high pressure liquid chromatography (HPLC). The encapsulation efficiency of Gln was determined to be the higher than the ones reported in the literature: 83.6%, 87.1% and 84.9% for 20 mM, 40 mM and 60 mM Gln, respectively. It was found that after 6 hours, liposomes loaded with 60 mM of Gln had released 45.7% of Gln. Optical microscopy images of the erythrocytes after 3 hours of incubation and haemolysis measurements proved that presence of liposomes did not cause any structural changes on the erythrocyte shape. Overall, it was concluded that L-Gln loaded PC/PG liposomes provide promising results in terms of developing a new drug delivery platform for SCD.


Assuntos
Glutamina , Lipossomos , Eritrócitos , Fosfatidilcolinas , Fosfatidilgliceróis
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