4 research outputs found
Model Lipid Raft Membranes for Embedding Integral Membrane Proteins: Reconstitution of HMG-CoA Reductase and Its Inhibition by Statins
For the first time, HMG-CoA reductase, the membrane protein
responsible
for cholesterol synthesis, was incorporated into a lipid membrane
consisting of DOPC:Chol:SM at a 1:1:1 molar ratio, which mimics the
lipid rafts of cell membranes. The membrane containing the protein
was generated in the form of either a proteoliposomes or a film obtained
by spreading the proteoliposomes at the airâwater interface
to prepare a protein-rich and stable lipid layer over time. The lipid
vesicle parameters were characterized using dynamic light scattering
(DLS) and fluorescence microscopy. The incorporation of HMG-CoA reductase
was reflected in the increased size of the proteoliposomes compared
to that of the empty liposomes of model rafts. Enzyme reconstitution
was confirmed by measuring the activity of NADPH, which participates
in the catalytic process. The thin lipid raft films formed by spreading
liposomes and proteoliposomes at the airâwater interface were
investigated using the Langmuir technique. The activities of the HMG-CoA
reductase films were preserved over time, and the two lipid raft systems,
nanoparticles and films, were exposed to solutions of fluvastatin,
a HMG-CoA reductase inhibitor commonly used in the treatment of hypercholesÂterolemia.
Both lipid raft systems constructed were useful membrane models for
the determination of reductase activity and for monitoring the statin
inhibitory effects and may be used for investigating other integral
membrane proteins during exposure to inhibitors/activators considered
to be potential drugs
Simvastatin Coadministration Modulates the Electrostatically Driven Incorporation of Doxorubicin into Model Lipid and Cell Membranes
Understanding the interactions between drugs and lipid
membranes
is a prerequisite for finding the optimal way to deliver drugs into
cells. Coadministration of statins and anticancer agents has been
reported to have a positive effect on anticancer therapy. In this
study, we elucidate the mechanism by which simvastatin (SIM) improves
the efficiency of biological membrane penetration by the chemotherapeutic
agent doxorubicin (DOX) in neutral and slightly acidic solutions.
The incorporation of DOX, SIM, or a combination of them (DOX:SIM)
into selected single-component lipid membranes, zwitterionic unsaturated
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), neutral cholesterol, and negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) was assessed
using the Langmuir method. The penetration of neutral lipid monolayers
by the codelivery of SIM and DOX was clearly facilitated at pH 5.5,
which resembles the pH conditions of the environment of cancer cells.
This effect was ascribed to partial neutralization of the DOX positive
charge as the result of intermolecular interactions between DOX and
SIM. On the other hand, the penetration of the negatively charged
DMPS monolayer was most efficient in the case of the positively charged
DOX. The efficiency of the drug delivery to the cell membranes was
evaluated under in vitro conditions using a panel
of cancer-derived cell lines (A172, T98G, and HeLa). MTS and trypan
blue exclusion assays were performed, followed by confocal microscopy
and spheroid culture tests. Cells were exposed to either free drugs
or drugs encapsulated in lipid carriers termed cubosomes. We demonstrated
that the viability of cancer cells exposed to DOX was significantly
impaired in the presence of SIM, and this phenomenon was greatly magnified
when DOX and SIM were coencapsulated in cubosomes. Overall, our results
confirmed the utility of the DOX:SIM combination delivery, which enhances
the interactions between neutral components of cell membranes and
positively charged chemotherapeutic agents
Monoolein Cubic Phase Gels and Cubosomes Doped with Magnetic NanoparticlesâHybrid Materials for Controlled Drug Release
Hybrid materials
consisting of a monoolein lipidic cubic phase
(LCP) incorporating two types of magnetic nanoparticles (NP) were
designed as addressable drug delivery systems. The materials, prepared
in the form of a gel, were subsequently used as a macroscopic layer
modifying an electrode and, after dispersion to nanoscale, as magnetocubosomes.
These two LCPs were characterized by small-angle X-ray scattering
(SAXS), cross-polarized microscopy, magnetic measurements, and phase
diagrams. The magnetic dopants were hydrophobic NP<sub>oleic</sub> and hydrophilic NP<sub>citric</sub>, characterized by dynamic light
scattering (DLS) and transmission electron microscopy (TEM), and their
influence on the properties of the cubic phases was investigated.
The removal of the anticancer drug, Doxorubicin (Dox) from the hybrid
cubic phase gels was studied by electrochemical methods. The advantages
of incorporating magnetic nanoparticles into the self-assembled lipid
liquid crystalline phases include the ability to address the cubic
phase nanoparticle containing large amounts of drug and to control
the kinetics of the drug release
Dependence of Interfacial Film Organization on Lipid Molecular Structure
Combination of surface analytical
techniques was employed to investigate
the interfacial behavior of the two designed lipidsî¸<i>N</i>-stearoylglycine (<b>1</b>) and its bulky neutral
headgroup-containing derivative <i>N</i>-stearoylvaline
ethyl ester (<b>2</b>)î¸at the airâsolution interface
and as transferred layers on different substrates. Formation of monolayers
at the airâwater interface was monitored on pure water and
on aqueous solutions of different pH. Crystallization effects were
visualized at pure water by recording the hystereses in the LangmuirâBlodgett
(LB) isotherms and by transferring the layers onto mica, gold (111),
and ITO (indiumâtin oxide on glass) electrodes. Subphase pH
affects the morphology and patch formation in monolayers of <b>1</b>, as evidenced by BAM measurements. At pH 8.2, formation
of well-ordered crystallites is observed, which upon compression elongate
according to predominantly 1-D growth mechanism to form a dense layer
of crystallites. This effect is not observed in monolayers of <b>2</b>, whose headgroup is not protonated. The orientation of layers
of <b>1</b> transferred to the solid supports is also pH dependent,
and their stability can be related to formation of a hydrogen-bonded
networks. AFM images of <b>1</b> exhibited platelets of multilayer
phase. The IR spectra of the ITO substrates covered by <b>1</b> indicated formation of hydrogen bonds between the amide groups.
The nature of the adsorption layer and its organization as a function
of potential were studied in-depth by EC STM using Au(111) as the
substrate. A model showing the arrangement of hydrogen bonds between
adsorbed molecules is presented and related to the observed organization
of the layer