The modulating effect of lipid bilayer/p-coumaric acid interactions on electrical properties of model lipid membranes and human glioblastoma cells.

Biological membranes are one of the most important elements of living cells determining their permeability to the active compounds. Still, little is known about the drug-membrane interactions in terms of pharmacological properties of potential drugs. Chemoprevention based on natural compounds is becoming a strong trend in modern oncopharmacology, and p-coumaric acid (p-CoA) is one such compound with tentative anticancer activity. The microelectrophoretic mobility measurements and electrochemical impedance spectroscopy were applied to study the effects of p-CoA on electrical properties of liposomes, spherical bilayers, and human glioblastoma cell membranes. Our results demonstrated that after treatment with p-CoA, the surface charge of LBC3, LN-229 and LN-18 cell lines was significantly changed in alkaline pH solutions, but not in acidic pH solutions. In contrast, no changes in surface charge density values were registered for phosphatidylethanolamine liposomal membranes and A172 cell membranes after p-CoA treatment. The impedance data showed an increase in values of both the electrical capacitance and the electrical resistance, indicating that p-CoA can be partially inserted into the phosphatidylcholine bilayers. The MTT assay showed cell line-dependent cytotoxic effect of p-CoA. Further molecular analyses revealed the ATP depletion and gene transcription modulation, which might indicate organelle membrane-crossing potential of p-CoA. These results suggest, that changes in surface charge of membranes of living cells not only might be potential predictor of membrane permeability, but also indicate differential composition of cell membranes in various cell lines. Thus further multidirectional analyses are required to implement electrochemical methods as standard testing procedures during drug development process.

Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers.

AIMS: We have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD). METHODS: Interaction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir-Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy. RESULTS: All dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive. CONCLUSIONS: HIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.

Custom AFM for X-ray beamlines: in situ biological investigations under physiological conditions.

A fast atomic force microscope (AFM) has been developed that can be installed as a sample holder for grazing-incidence X-ray experiments at solid/gas or solid/liquid interfaces. It allows a wide range of possible investigations, including soft and biological samples under physiological conditions (hydrated specimens). The structural information obtained using the X-rays is combined with the data gathered with the AFM (morphology and mechanical properties), providing a unique characterization of the specimen and its dynamics in situ during an experiment. In this work, lipid monolayers and bilayers in air or liquid environment have been investigated by means of AFM, both with imaging and force spectroscopy, and X-ray reflectivity. In addition, this combination allows the radiation damage induced by the beam on the sample to be studied, as has been observed on DOPC and DPPC supported lipid bilayers under physiological conditions.

Biopharmaceutical studies of a novel sedative sublingual lozenge based on glycine and tryptophan: A rationale for mucoadhesive agent selection.

Effective sedative drugs are in great demand due to increasing incidence of nervous disorders. The present work was aimed to develop a novel sublingual sedative drug based on glycine and L-tryptophan amino acids. Carbopol and different hydroxypropyl methylcellulose species were alternatively tested as mucoadhesive agents intended to prolong tryptophan sublingual release time. A model lipid medium of fully hydrated L-α-dimyristoylphosphatidylcholine was used for optimal mucoadhesive agents selection. Simultaneous processes of drug release and diffusion in lipid medium were first investigated involving both experimental and theoretical approaches. Individual substances, their selected combinations as well as different drug formulations were consecutively examined. Application of kinetic differential scanning calorimetry method allowed us to reveal a number of specific drug-excipient effects. Lactose was found to essentially facilitate tryptophan release and provide its ability to get into the bloodstream simultaneously with glycine, which is necessary to achieve glycine-tryptophan synergism. Introduction of a mucoadhesive agent into the formulation was shown to change kinetics of drug-membrane interactions variously depending on viscosity grade. Among the mucoadhesive agents, hydroxypropyl methylcellulose species K4M and E4M were shown to further accelerate drug release, therefore they were selected as optimal. Thus, effectiveness of the novel sedative drug was provided by including some excipients, such as lactose and the selected mucoadhesive agent species. A dynamic mathematical model was developed properly describing release and diffusion in lipid medium of various drug substances. Our study clearly showed applicability of a lipid medium to meet challenges such as drug-excipient interactions and optimization of drug formulations.

Incorporation of simvastatin into lipid membranes: Why deliver a statin in form of inclusion complex with hydrophilic cyclodextrin.

In this work, the effects of simvastatin (SIM), (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) and their complex (SIM:HPßCD) on the structure and properties of lipid membranes were investigated for the first time by Langmuir technique combined with PM-IRRAS spectroscopy. An improved understanding of the differences of the interactions between free SIM, and SIM in the form of an inclusion complex with HPßCD with the lipid membrane will improve the development of preparation methods for in vivo applications. Monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol) and their mixture DMPC:Chol (7:3) served as simple models of one leaflet of the cell membrane. The penetration of well-organized lipid layers by simvastatin lead to their fluidization but the extent of this unwanted effect was smaller when the drug was delivered in the form of the SIM:HPßCD complex. Surface pressure vs. time dependencies showed that the drug encapsulated with cyclodextrin dissociated from the complex upon contact with the lipid layer and the weak interactions between the exterior polar part of the HPßCD and the polar headgroups of the lipid layer facilitated smooth incorporation of the released lipophilic drug into the membrane. At a longer time-scale, the HPßCD ligand released from the complex removed some cholesterol, but not DMPC, from the lipid layer, hence, similarly to the enzyme inhibiting action of statins - it lead to the decrease of the amount of cholesterol in the membrane. Delivery of simvastatin in the form of an inclusion complex with HPßCD is proposed as an approach improving its bioavailability in the cholesterol-lowering therapies.

Glucose-modified carbosilane dendrimers: Interaction with model membranes and human serum albumin.

Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM2-3Glu, significantly affect the type and extent of interaction with biological structures.

Synthesis, characterization, and interactions of single-walled carbon nanotubes modified with doxorubicin with Langmuir-Blodgett biomimetic membranes.

The synthesis, characterization, and the influence of single-walled carbon nanotubes (SWCNTs) modified with an anticancer drug doxorubicin (DOx) on the properties of model biological membrane as well as the comparison of the two modes of modification has been presented. The drug was covalently attached to the nanotubes either preferentially on the sides or at the ends of the nanotubes by the formation of hydrazone bond. The efficiency of the modification was proved by the results of FTIR, Raman, and thermogravimetric analysis. In order to characterize the influence of SWCNT-DOx conjugates on model biological membranes, Langmuir technique has been employed. The mixed monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and SWCNT-DOx with different weight ratio have been prepared. It has been shown that changes in the isotherm characteristics depend on the SWCNTs content. While smaller amounts of SWCNTs do not exert significant differences, the introduction of the prevailing content of the nanotubes increases area per molecule and decreases the maximum value of compression modulus, leading to more fluid monolayer. However, upon increasing the surface pressure, the aggregation of carbon nanotubes within the thiolipid matrix has been observed. Mixed layers of DPPTE/SWCNT-DOx were also transferred onto gold electrodes by means of LB method. Cyclic voltammetry showed that SWCNT-DOx conjugates remain adsorbed at the electrode surface and are stable in time. Additionally, higher values of peak current and DOx surface concentration obtained for side modification prove that side modification allows for more efficient conjugation of the drug to carbon nanotubes. Graphical abstractᅟ.

Short periodicity phase based on ceramide [AP] in the model lipid membranes of stratum corneum does not change during hydration.

Small angle X-ray scattering technique was used to determine electron density profiles of short periodicity phase in the model lipid membranes of stratum corneum at different pH. Basic quaternary system was prepared as used previously in the neutron experiments at partial hydration. It was shown that electron density profiles of partially hydrated and fully hydrated model lipid membranes with four basic components were quite similar and demonstrated almost no interbilayer water.

Comparison of the interactions of daunorubicin in a free form and attached to single-walled carbon nanotubes with model lipid membranes.

In this work the interactions of an anticancer drug daunorubicin (DNR) with model thiolipid layers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) were investigated using Langmuir technique. The results obtained for a free drug were compared with the results recorded for DNR attached to SWCNTs as potential drug carrier. Langmuir studies of mixed DPPTE-SWCNTs-DNR monolayers showed that even at the highest investigated content of the nanotubes in the monolayer, the changes in the properties of DPPTE model membranes were not as significant as in case of the incorporation of a free drug, which resulted in a significant increase in the area per molecule and fluidization of the thiolipid layer. The presence of SWCNTs-DNR in the DPPTE monolayer at the air-water interface did not change the organization of the lipid molecules to such extent as the free drug, which may be explained by different types of interactions playing crucial role in these two types of systems. In the case of the interactions of free DNR the electrostatic attraction between positively charged drug and negatively charged DPPTE monolayer play the most important role, while in the case of SWCNTs-DNR adducts the hydrophobic interactions between nanotubes and acyl chains of the lipid seem to be prevailing. Electrochemical studies performed for supported model membranes containing the drug delivered in the two investigated forms revealed that the surface concentration of the drug-nanotube adduct in supported monolayers is comparable to the reported surface concentration of the free DNR incorporated into DPPTE monolayers on gold electrodes. Therefore, it may be concluded that the application of carbon nanotubes as potential DNR carrier allows for the incorporation of comparable amount of the drug into model membranes with simultaneous decrease in the negative changes in the membrane structure and organization, which is an important aspect in terms of side effects of the drug.