Exploring the interactions of irbesartan and irbesartan–2-hydroxypropyl-β-cyclodextrin complex with model membranes

The interactions of irbesartan (IRB) and irbesartan–2-hydroxypropyl-β-cyclodextrin (HP-β-CD) complex with Dipalmitoyl Phosphatidylcholine (DPPC) bilayers have been explored utilizing an array of biophysical techniques ranging from Differential Scanning Calorimetry (DSC), Small angle X-ray Scattering (SAXS), ESI Mass-Spectrometry (ESI-MS) and solid state Nuclear Magnetic Resonance (ssNMR). Molecular Dynamics (MD) calculations have been also conducted to complement the experimental results. Irbesartan was found to be embedded in the lipid membrane core and to affect the phase transition properties of the DPPC bilayers. SAXS studies revealed that irbesartan alone does not display perfect solvation since some coexisting irbesartan crystallites are present. In its complexed form IRB gets fully solvated in the membranes showing that encapsulation of IRB in HP-β-CD may have beneficial effects in the ADME properties of this drug. MD experiments revealed the topological and orientational integration of irbesartan into the phospholipid bilayer being placed at about 1 nm from the membrane centre

The Insertion and Transport of Anandamide in Synthetic Lipid Membranes Are Both Cholesterol-Dependent

International audienceBackground: Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide.Methodology/Principal Findings: Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide.Conclusions/Significance: Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications

Exploring the role of cyclodextrins as a cholesterol scavenger: a molecular dynamics investigation of conformational changes and thermodynamics

This study presents a comprehensive analysis of the cholesterol binding mechanism and conformational changes in cyclodextrin (CD) carriers, namely βCD, 2HPβCD, and MβCD. The results revealed that the binding of cholesterol to CDs was spontaneous and thermodynamically favorable, with van der Waals interactions playing a dominant role, while Coulombic interactions have a negligible contribution. The solubility of cholesterol/βCD and cholesterol/MβCD complexes was lower compared to cholesterol/2HPβCD complex due to stronger vdW and Coulombic repulsion between water and CDs. Hydrogen bonding was found to have a minor role in the binding process. The investigation of mechanisms and kinetics of binding demonstrated that cholesterol permeates into the CD cavities completely. Replicas consideration indicated that while the binding to 2HPβCD occurred perpendicularly and solely through positioning cholesterol's oxygen toward the primary hydroxyl rim (PHR), the mechanism of cholesterol binding to βCD and MβCD could take place with the orientation of oxygen towards both rims. Functionalization resulted in decreased cavity polarity, increased constriction tendency, and altered solubility and configuration of the carrier. Upon cholesterol binding, the CDs expanded, increasing the cavity volume in cholesterol-containing systems. The effects of cholesterol on the relative shape anisotropy (κ 2) and asphericity parameter (b) in cyclodextrins were investigated. βCD exhibited a spherical structure regardless of cholesterol presence, while 2HPβCD and MβCD displayed more pronounced non-sphericity in the absence of cholesterol. Loading cholesterol transformed 2HPβCD and MβCD into more spherical shapes, with increased probabilities of higher κ 2. MβCD showed a higher maximum peak of κ 2 compared to 2HPβCD after cholesterol loading, while 2HPβCD maintained a significant maximum peak at 0.2 for b

The use of molecular dynamics simulation for the study of polymeric and lipid based drug delivery systems

Systemic administration is the conventional method for administrating drugs. Following injection, ideally, we wish the drug only to locate to the target tissue, however, this is not what occurs; the drug molecules rather distribute throughout the entire body via the blood stream. Regarding some drugs, in particular chemotherapy agents, this often leads to severe dose limiting side effects and unsatisfactory therapeutic results. On the other hand, many drugs as is also the case for the chemotherapy agents, demonstrate low aqueous solubility and suboptimal pharmacokinetic properties. These problems all necessitate the use of drug delivery systems (DDSs) as they decrease the side effects of drugs while also improving drug bioavailability and pharmacokinetics. Although there are different varieties of DDSs, we have focused on those categorized as polymeric or lipidic. Depending on the drug to be delivered and site of action of the drug, polymeric DDSs can be used either locally or systemically. Hydrogels and electrospun polymer fibers are two examples of polymeric DDSs that are used for the local delivery of many drugs, including antibiotics and anticancer drugs. The other form of polymeric DDSs are nanoparticles that are capable of carrying and in some cases targeting drug molecules. These polymeric DDSs are generally injected into the blood stream to reach their target site. Lipidic DDSs mainly are used in the form of nanoparticles that, depending on their lipid composition and method of preparation, would have different characteristics. Liposomes and solid lipid nanoparticles are two examples of lipidic DDSs. Despite the huge number of publications regarding the use of nanoparticles as DDSs, the number of approved drug therapies that make use of nanoparticle-based delivery systems still remains small. One of the reasons for this problem is that formulations of DDSs are complicated and difficult to optimize. Drug delivery systems should be further redesigned and optimized, however, this has proved challenging due to intrinsic and practical experimental limitations. For example, it is difficult to experimentally elucidate the reason many DDSs show promise in vitro but fail in vivo. The limitations to the extent to which mechanistic insight can be gained from experiments regarding DDSs can be compensated by computational molecular modelling techniques that provide detailed information on molecular interactions of drugs and carriers. The insights obtained by the studies performed in this thesis can be used to improve the design of DDSs. In this thesis, two polymeric (studies I and IV) and two lipidic (studies II and III) DDSs were studied by all-atom molecular dynamics (MD) simulations. In each of these studies, a specific property of the DDS was evaluated in detail. These properties are drug release profile (study I), stability (study II), pH-sensitivity (study III) and size (study IV). We evaluated these properties through investigation of the three varieties of interactions DDSs have: interactions of DDSs with the loaded drug, interactions among the components of DDSs and interactions between the DDSs and the medium, namely water and ions. While it is difficult to directly determine an accurate picture of these interactions experimentally at atomic scale resolution, all- atom MD simulation can provide insight into this.Lääkeaineet annostellaan yleensä systeemisesti ja olisi ideaalista, että annostelun jälkeen lääkeaine vaikuttaisi vain paikallisesti kohdekudoksessa. Käytännössä näin ei kuitenkaan tapahdu, vaan pikemminkin lääkeainemolekyylit jakautuvat koko kehoon verenkierron mukana. Joidenkin lääkkeiden, erityisesti kemoterapeuttisten aineiden kohdalla, tämä johtaa usein vakaviin annosta rajoittaviin sivuvaikutuksiin ja näin ollen epätyydyttäviin terapeuttisiin tuloksiin. Toisaalta monilla lääkkeillä, kuten myös kemoterapia-aineilla, on myös alhainen vesiliukoisuus ja huonot farmakokineettiset ominaisuudet. Kaikki nämä ongelmat edellyttävät erilaisten lääkekuljetusjärjestelmien käyttöä, koska ne vähentävät esimerkiksi haitallisia sivuvaikutuksia ja parantavat lääkeaineiden biologista hyötyosuutta. Vaikka lääkekuljetusjärjestelmiä on erilaisia, olemme keskittyneet tässä väitöskirjassa vain niihin, jotka on luokiteltu polymeeri- tai lipidipohjaisiksi. Kuljetettavasta lääkeaineesta ja lääkkeen vaikutuspaikasta riippuen polymeeripohjaisia lääkekuljetusjärjestelmiä voidaan käyttää paikallisesti tai systeemisesti. Hydrogeelit ja sähkökehrätyt polymeerikalvot ovat esimerkkejä tällaisista lääkekuljetusjärjestelmistä ja niitä käytetään monien lääkkeiden, kuten antibioottien ja syöpälääkkeiden paikalliseen annosteluun. Polymeeripohjaiset nanohiukkaset pystyvät vuorostaan kuljettamaan ja joissakin tapauksissa myös kohdentamaan lääkeainemolekyylejä. Nanohiukkaset ruiskutetaan yleensä suoraan verenkiertoon, jotta ne saavuttaisivat terapeuttisen kohteen. Lipideistä koostuvat lääkekuljetusjärjestelmät ovat pääasiassa nanohiukkasia, joilla on lipidikoostumuksesta ja valmistusmenetelmästä johtuen erilaisia ominaisuuksia. Liposomit ja kiinteät lipidinanohiukkaset ovat esimerkkejä lääkeaineiden kuljetusjärjestelmistä, jotka pohjautuvat rasva-aineisiin eli lipideihin. Siitä huolimatta, että kirjallisuudesta löytyy valtava määrä tieteellisiä julkaisuja, jotka liittyvät nanohiukkasten käyttöön lääkekuljetusjärjestelminä, hyväksyttyjen nanohiukkaspohjaisten lääkehoitomuotojen määrä on edelleen pieni. Tämä johtuu siitä, että valmisteet ovat monimutkaisia, vaikeasti optimoitavissa. Nanohiukkasia tulisi edelleen suunnitella ja optimoida, mutta tämä on osoittautunut haastavaksi mittalaitteiden rajoituksien vuoksi. Esimerkiksi on erittäin vaikeaa selvittää kokeellisesti, miksi monet nanohiukkaset ovat lupaavia in vitro mittauksissa, mutta epäonnistuvat in vivo kokeissa. Kokeellisia mittauksia, joissa nanohiukkasista saadaan mekanistista tietoa, voidaan kompensoida erilaisilla in silico molekyylimallinnustekniikoilla, jotka tarjoavat yksityiskohtaista tietoa lääkeaineiden ja kantajien molekyylivuorovaikutuksista. Väitöskirjassa esitettyjä tuloksia voidaan hyödyntää lääkeaineiden kuljetusjärjestelmien suunnittelussa. Tässä väitöskirjassa tutkittiin kahta polymeereistä (tutkimukset I ja IV) ja kahta lipideistä (tutkimukset II ja III) koostuvaa lääkekuljetusjärjestelmää hyödyntäen atomistisia molekyylidynamiikka simulaatioita. Jokaisessa tutkimuksessa lääkekuljetusjärjestelmän tietty ominaisuus arvioitiin yksityiskohtaisesti. Näitä ominaisuuksia olivat lääkeaineen vapautumisprofiili (tutkimus I), stabiilius (tutkimus II), pH-herkkyys (tutkimus III) ja koko (tutkimus IV). Arvioimme näitä ominaisuuksia tutkimalla kolmea erilaista vuorovaikutusta lääkekuljetusjärjestelmissä: matriisin vuorovaikutus ladatun lääkkeen kanssa, matriisin eri komponenttien vuorovaikutus keskenään sekä vuorovaikutus lääkekuljetusjärjestelmän ja väliaineen (vesi ja ionit) välillä. Siitä huolimatta, että kokeellisilla mittauksilla on erittäin vaikeaa tuottaa atomitason kuva näistä vuorovaikutuksista, voidaan näistä saada laskennallisia molekyylidynamiikkasimulaatioita hyödyntäen hyvä käsitys

Hepatocyte cholesterol content modulates glucagon receptor signalling

Objective To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels. Methods We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content. Results GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator. Conclusions Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance