DPPC
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Liposomal carrier systems have emerged as a promising technology for pulmonary drug delivery. This study focuses on two selected liposomal systems, namely, dipalmitoylphosphatidylcholine stabilized by phosphatidic acid and cholesterol (DPPC-PA-Chol) and dipalmitoylphosphatidylcholine stabilized by polyethylene glycol and cholesterol (DPPC-PEG-Chol). First, the research investigates the stability of these liposomal systems during the atomization process using different kinds of nebulizers (air-jet, vibrating mesh, and ultrasonic). The study further explores the aerodynamic particle size distribution of the aerosol generated by the nebulizers. The nebulizer that demonstrated optimal stability and particle size was selected for more detailed investigation, including Andersen cascade impactor measurements, an assessment of the influence of flow rate and breathing profiles on aerosol particle size, and an in vitro deposition study on a realistic replica of the upper airways. The most suitable combination of a nebulizer and liposomal system was DPPC-PA-Chol nebulized by a Pari LC Sprint Star in terms of stability and particle size. The influence of the inspiration flow rate on the particle size was not very strong but was not negligible either (decrease of Dv50 by 1.34 μm with the flow rate increase from 8 to 60 L/min). A similar effect was observed for realistic transient inhalation. According to the in vitro deposition measurement, approximately 90% and 70% of the aerosol penetrated downstream of the trachea using the stationary flow rate and the realistic breathing profile, respectively. These data provide an image of the potential applicability of liposomal carrier systems for nebulizer therapy. Regional lung drug deposition is patient-specific; therefore, deposition results might vary for different airway geometries. However, deposition measurement with realistic boundary conditions (airway geometry, breathing profile) brings a more realistic image of the drug delivery by the selected technology. Our results show how much data from cascade impactor testing or estimates from the fine fraction concept differ from those of a more realistic case.
- MeSH
- 1,2-dipalmitoylfosfatidylcholin MeSH
- aerosoly MeSH
- aplikace inhalační MeSH
- bronchodilatancia * MeSH
- cholesterol MeSH
- design vybavení MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- liposomy MeSH
- nebulizátory a vaporizátory MeSH
- trachea * MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pulmonary surfactant is a complex mixture of lipids and proteins lining the interior of the alveoli, and constitutes the first barrier to both oxygen and pathogens as they progress toward blood circulation. Despite decades of study, the behavior of the pulmonary surfactant at the molecular scale is poorly understood, which hinders the development of effective surfactant replacement therapies, useful in the treatment of several lung-related diseases. In this work, we combined all-atom molecular dynamics simulations, Langmuir trough measurements, and AFM imaging to study synthetic four-component lipid monolayers designed to model protein-free pulmonary surfactant. We characterized the structural and dynamic properties of the monolayers with a special focus on lateral heterogeneity. Remarkably, simulations reproduce almost quantitatively the experimental data on pressure-area isotherms and the presence of lateral heterogeneities highlighted by AFM. Quite surprisingly, the pressure-area isotherms do not show a plateau region, despite the presence of liquid-condensed nanometer-sized domains at surface pressures larger than 20 mN/m. In the simulations, the liquid-condensed domains were small and transient, but they did not coalesce to yield a separate phase. They were only slightly enriched in DPPC and cholesterol, and their chemical composition remained very similar to the overall composition of the monolayer membrane. Instead, they differed from liquid-expanded regions in terms of membrane thickness (in agreement with AFM data), diffusion rates, as well as acyl chain packing and orientation. We hypothesize that such lateral heterogeneities are crucial for lung surfactant function, as they allow both efficient packing, to achieve low surface tension, and sufficient fluidity, critical for rapid adsorption to the air-liquid interface during the breathing cycle.
- Publikační typ
- časopisecké články MeSH
The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.
- MeSH
- cholesterol chemie MeSH
- difenylhexatrien chemie MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční polarizace MeSH
- lipidové dvojvrstvy chemie MeSH
- molekulární konformace MeSH
- sfingomyeliny chemie MeSH
- simulace molekulární dynamiky MeSH
- tranzitní teplota MeSH
- vztahy mezi strukturou a aktivitou MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
Our study demonstrates that nanoplasmonic sensing (NPS) can be utilized for the determination of the phase transition temperature (Tm) of phospholipids. During the phase transition, the lipid bilayer undergoes a conformational change. Therefore, it is presumed that the Tm of phospholipids can be determined by detecting conformational changes in liposomes. The studied lipids included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Liposomes in gel phase are immobilized onto silicon dioxide sensors and the sensor cell temperature is increased until passing the Tm of the lipid. The results show that, when the system temperature approaches the Tm, a drop of the NPS signal is observed. The breakpoints in the temperatures are 22.5 °C, 41.0 °C, and 55.5 °C for DMPC, DPPC, and DSPC, respectively. These values are very close to the theoretical Tm values, i.e., 24 °C, 41.4 °C, and 55 °C for DMPC, DPPC, and DSPC, respectively. Our studies prove that the NPS methodology is a simple and valuable tool for the determination of the Tm of phospholipids.
Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.
- MeSH
- 1,2-dipalmitoylfosfatidylcholin chemie MeSH
- 2-naftylamin analogy a deriváty chemie MeSH
- chemické modely * MeSH
- fluorescence MeSH
- fosfatidylcholiny chemie MeSH
- kvantová teorie MeSH
- laurany chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- simulace molekulární dynamiky * MeSH
- Publikační typ
- časopisecké články MeSH
The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčná membrána metabolismus MeSH
- cytoplazma metabolismus MeSH
- fluorescenční barviva metabolismus MeSH
- kinetika MeSH
- mutageneze cílená metody MeSH
- simulace molekulového dockingu MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- tryptofan metabolismus MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Structural properties of plasmid DNA and model lipid membrane treated with newly synthesized platinum(II) complex cis-[PtCl2{P(CH2CH2COOH)3}2] (cis-DTCEP for short) were studied and compared with effects of anticancer drug cisplatin, cis-[Pt(NH3)2Cl2] (cis-DDP for short). Time Correlated Single Photon Counting Fluorescence Correlation Spectroscopy (TCSPC-FCS) was employed to study interactions between those platinum complexes and DNA. The TCSPC-FCS results suggest that bonding of cis-DTCEP derivative to DNA leads to plasmid strain realignment towards much more compact structure than in the case of cis-DDP. Application of both differential scanning calorimetry and infrared spectroscopy to platinum complexes/DPPC showed that cis-DTCEP slightly increases the phospholipid's main phase transition temperature resulting in decreased fluidity of the model membrane. The newly investigated compound-similarly to cis-DDP-interacts mainly with the DPPC head group however not only by the means of electrostatic forces: this compound probably enters into hydrophilic region of the lipid bilayer and forms hydrogen bonds with COO groups of glycerol and PO2- group of DPPC.
Biological membranes generate specific functions through compartmentalized regions such as cholesterol-enriched membrane nanodomains that host selected proteins. Despite the biological significance of nanodomains, details on their structure remain elusive. They cannot be observed via microscopic experimental techniques due to their small size, yet there is also a lack of atomistic simulation models able to describe spontaneous nanodomain formation in sufficiently simple but biologically relevant complex membranes. Here we use atomistic simulations to consider a binary mixture of saturated dipalmitoylphosphatidylcholine and cholesterol - the "minimal standard" for nanodomain formation. The simulations reveal how cholesterol drives the formation of fluid cholesterol-rich nanodomains hosting hexagonally packed cholesterol-poor lipid nanoclusters, both of which show registration between the membrane leaflets. The complex nanodomain substructure forms when cholesterol positions itself in the domain boundary region. Here cholesterol can also readily flip-flop across the membrane. Most importantly, replacing cholesterol with a sterol characterized by a less asymmetric ring region impairs the emergence of nanodomains. The model considered explains a plethora of controversial experimental results and provides an excellent basis for further computational studies on nanodomains. Furthermore, the results highlight the role of cholesterol as a key player in the modulation of nanodomains for membrane protein function.
Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Nε-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.
- MeSH
- guanidiny farmakologie MeSH
- hmotnostní spektrometrie MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- ledviny metabolismus MeSH
- ouabain farmakologie MeSH
- oxidační stres MeSH
- produkty pokročilé glykace chemie metabolismus MeSH
- pyruvaldehyd chemie metabolismus MeSH
- sérový albumin hovězí metabolismus MeSH
- skot MeSH
- sodíko-draslíková ATPasa antagonisté a inhibitory chemie metabolismus MeSH
- Sus scrofa MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
New butyltin complexes with 2-sulfobenzoic acid: [Sn(C4H9)2{O3SC6H4COO-2}(H2O)]·(C2H5OH) (DBTsbz), [Sn(C4H9)3{O3SC6H4COOH-2}] (TBTsbz) and [Sn2(C4H9)6{μ-O3SC6H4COO-2}] (DTBTsbz) are very effective cytotoxic agents against tumor cells. The molecular interaction of these complexes with lipid membranes and DNA has been investigated. The IR spectra and changes of (1)H, (13)C chemical shifts suggest that SO3 and COO groups of 2-sulfobenzoato ligand interact with O atom of glycerin fragment of DPPC. Moreover, the compounds form Sn-OP bonds with phosphate groups of DPPC, which was shown by the lower frequency shift of the νs(PO2(-)) and νas(PO2(-)) band, by change of (31)P NMR signals and by DFT calculation. Another possibility is the interaction of the phosphate group of DPPC owing to formation of hydrogen bond O-H…O-P between water molecule coordinated to Sn and oxygen atom from the phosphate group. Using TCSPC-FCS we characterized DNA supramolecular assemblies' formation upon increasing TBTsbz, DTBTsbz and DBTsbz concentration. Diffusion time, lifetime and particle number changes are altered systematically with increasing Ccomp/CDNAbp ratio in following effectiveness order DBTsbz > TBTsbz > DTBTsbz. From those parameters we can conclude that all these compounds lead to a change of DNA winding, strand but not to DNA compaction. Investigated compounds show very high cytotoxic activity against cancer cell lines. All compounds exhibit efficient in vitro antitumor activity toward Jurkat (T-cell leukemia), CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line) and D-17 (canine osteosarcoma). The DBTsbz is more effective then carboplatin against canine osteosarcoma.
- MeSH
- benzensulfonáty chemie farmakologie MeSH
- benzoáty chemie farmakologie MeSH
- DNA chemie metabolismus MeSH
- konformace nukleové kyseliny účinky léků MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie MeSH
- organocínové sloučeniny chemie farmakologie MeSH
- protinádorové látky chemie farmakologie MeSH
- screeningové testy protinádorových léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
