Vesicular Bilayer Research Articles

Comparative evaluation of pharmacokinetic parameters between a pure nisoldipine suspension and a nisoldipine-loaded bilosome suspension

Bilosomes are nanocarriers that contain bile salts in their vesicular bilayer, thereby enhancing their flexibility and durability in the gastrointestinal tract. Unlike conventional vesicular systems they provide distinct advantages such as streamlined manufacturing procedures, cost efficiency, and improved stability. The main objective of this study was to attain a comparison of the pharmacokinetic parameters of nisoldipine (NSD) after administering an ordinary NSD suspension and an NSD-loaded bilosome suspension. The study used 60 Swiss albino rats weighing 200±15 g and divided into two groups (n=30 each). A dose of 2.2 mg/kg of NSD was administered from the ordinary NSD suspension to the rats of the first group and the same dose of NSD-loaded bilosome suspension was administered to the rats of the second group. NSD levels were determined in the rat plasma by using high-performance liquid chromatography. Our results showed that the Cmax, the Tmax, and the AUC0-36 were 51.47±0.94 ng/mL, 2±0.3 h, and 323.33±21 ng×h/mL for the pure suspension, and 116.41±1.22 ng/mL, 4±0.7 h, and 916±64.09 ng×h/mL for the bilosome suspension, respectively. The maximum concentration was significantly different between the pure and the bilosomal preparation (P<0.05), while the relative bioavailability of the pure suspension was 2.9 times that of the bilosomal suspension, 36 h after a single-dose NSD administration. In conclusion, the prepared bilosomal suspension enhanced the bioavaila¬bility of NSD, and could be considered as a vital delivery system.

Fabrication of Microalgae Oil Vesicles for Drug Delivery Applications.

In this study, it is demonstrated that natural microalgae oils, which contain fatty acid components including docosahexaenoic acid (DHA), could be directly applied to fabricate vesicular structures in aqueous phase through a forced formation process. The microalgae oil vesicles had initial average diameters of 170- 230 nm with negative charges apparently caused by dissociation of the fatty acid components. The vesicles possessed excellent stability with lifetimes for at least 450 days. The formation of the vesicular structures with hydrophilic cores/regions was confirmed by the transmission electron microscopy (TEM) image and successful encapsulation of a hydrophilic material. For encapsulation of a hydrophobic material, lutein, the vesicle size was increased probably due to the insertion of lutein into the hydrophobic vesicular bilayer structures. The analysis of Fourier transform infrared (FTIR) spectroscopy suggested that the vesicular bilayer fluidity was decreased by encapsulating lutein. However, the lutein-encapsulating microalgae oil vesicles still possessed high stability and the vesicular structures could maintain intact even at an environmental temperature up to 60℃. Applicability of the microalgae oil vesicles as drug delivery carriers was also demonstrated by successful encapsulation of curcumin. However, when the loaded curcumin was increased to a certain amount, physical stability of the microalgae oil vesicles was significantly reduced. This is probably because the vesicular structures with only limited spaces for accommodating hydrophobic materials were strongly affected by encapsulating a large amount of curcumin. It is interesting to note that by adding egg L-α-phosphatidylcholine, the curcumin encapsulation-induced instability of the microalgae oil vesicles could be alleviated. The results indicated that vesicular structures could be fabricated from microalgae oils and the microalgae oil vesicles were capable of encapsulating hydrophilic or hydrophobic materials for drug delivery applications. The findings lay a background for further dosage form development of nutritional supplements encapsulated by natural microalgae oils.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Tailoring Niosomes- Implications for Controlled Cargo Release and Function as Nanoreactors.

Nonionic surfactant vesicles (Niosomes) were prepared using polyoxyethylene alkyl ether (Brij 58).The impact of variation of the Brij: cholesterol molar ratio on the niosomal structure was studied. Fluorescence studies performed with the membrane probe 1,6-Diphenyl-1,3,5-triene (DPH) gave important insight on the bilayer integrity of the niosomes in response to environmental perturbations. The aim of the work being assessment of the efficacy of the niosomes as "drug release vehicles", release studies were performed with a xanthene dye Carboxyfluorescein (CF). Further, the vesicles were used as nanoreactors for the synthesis of gold nanoparticles (GNPs) as it is often useful to house nanoparticles in biological /biomimicking environments. Stable, spherical GNPs of diameter 6-10 nm were formed in these vesicles. As the vesicular bilayer mimics the cell membrane, the present work is relevant to the use of the GNPs for diagnostic and therapeutic purpose. It has also been established that fluorescence resonance energy transfer (FRET) effectively occurs between DPH and CF in the niosomes. The FRET studies provide important insight on the location of dyes within the vesicles thus indicating the prospective applications of this fluorescence technique for tracking the location of probes in biomimicking systems which maybe extrapolated to in vivo biological systems in future.

Management and control of intraocular pressure applying macitentan hydrogel film formulation: improved effect of surfactant and cosurfactant system.

Macitentan blocks endothelin receptors in order to control the pulmonary arterial hypertension (PAH). Oral administration of macitentan is associated with painful urination and troubled breathing. Formulated macitentan hydrogel film was used for examining the control of intraocular pressure, and the effect of surfactant and cosurfactant was studied. Macitentan ocular film formulation has been prepared in hydroxypropyl methylcellulose (HPMC) matrix system using different surfactant/co-surfactant system, and intraocular pressure was monitored on normotensive rabbit eyes after application in the cul-de-sac. The solid state characterization of the film indicated amorphisation of macitentan and no issues regarding major incompatibility was observed. Combination of surfactant, co-surfactant and hydrophilic co-solvent systems in the said films markedly improved the drug release and mucosal tissue permeation. Presence of PEG and Transcutol significantly improved ex vivo corneal permeation of MP and MT respectively compared to other films. Transcutol (MT) exhibited greatest difference among the formulations by improving the vesicular bilayer fluidity and reducing the mucosal tissue barrier facilitating the transcorneal diffusion. A combination of diffusion and erosion control behavior was observed in drug release and corneal permeation of the films due to the balanced liquid penetration and polymeric chain relaxation rate. MP and MT films were used for further in vivo studies to achieve possible effective and prolonged control of intraocular pressure. In vivo study has revealed the reduction in intraocular pressure upto about 23 % when tested on normotensive rabbit model. The films has managed to lower the IOP upto 3 h. Developed macitentan hydrogel film containing Transcutol (MT) could have a high potential for the control and management of ocular hypertension after topical application.

A vesicle-to-sponge transition via the proliferation of membrane-linking pores in ω-3 polyunsaturated fatty acid-containing lipid assemblies

We investigate the nanostructure evolution and the membrane reorganization of diluted lipid dispersions of self-assembled monoolein (MO)/eicosapentaenoic acid (EPA, 20:5) mixtures by synchrotron small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) microscopy. The nonlamellar lipid phase containing a ω-3 polyunsaturated fatty acid was fragmented into stable nanoscale objects with the help of PEGylated lipids. The Cryo-TEM imaging revealed the transformation pathway of the vesicular bilayer membranes into sponge nanoparticles (spongosomes) in excess aqueous medium. At ambient temperature, the topological transition occurred through the proliferation of membrane-linking pores (MLP) within the individual lipid nanoparticles. The density of the MLP pores varied starting from the nanoparticle center toward the periphery. The generation of MLP is governed by the amphiphilic composition and leads to formation of 3D networks of aqueous channels inside the nanoparticles, i.e. spongosomes. A higher density of MLP pores was established at increasing fraction of EPA in the mixed lipid membranes. This corresponded to sponge particles of less hydrated internal structure, i.e. with smaller-size aqueous compartments. Synchrotron SAXS patterns characterized the overall structural transition from vesicles to sponge membranes in the studied lipid systems. It can be concluded that the incorporation of a ω-3 polyunsaturated fatty acid at increasing concentration causes swelling inhibition (dehydration) of the host liquid crystalline architectures.

Enhanced physical stability of positively charged catanionic vesicles: Role of cholesterol-adjusted molecular packing

Abstract A pseudodouble-chain ion pair amphiphile, hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), and a double-chain cationic surfactant, ditetradecyldimethylammonium bromide (DTDAB), were used as the main materials to form positively charged catanionic vesicles with various cholesterol contents. The effects of cholesterol on physical stability of the charged catanionic vesicles were then investigated by size, zeta potential, and Fourier transform infrared analyses. With the presence of cholesterol, the physical stability of the vesicles was enhanced. The inclusion of cholesterol would increase the distance between the charged headgroups of the vesicle-forming molecules and reduce the tendency of the counterion binding onto the charged vesicle surfaces, leading to a more pronounced charge character of the vesicles. Furthermore, with the incorporation of cholesterol, the sterol ring of cholesterol tended to maximize the contact with neighboring hydrocarbon chains, thus improving the bilayer mechanical strength. In the meantime, the alkyl side-chain of cholesterol together with the segments of the hydrocarbon chains near the ends in the vesicle-forming molecules could form flexible regions within the vesicular bilayers. Thus the presence of cholesterol in the bilayer structures of HTMA-DS/DTDAB catanionic vesicles not only enhanced the inter-vesicular electrostatic repulsion but also adjusted the intra-vesicular molecular packing, resulting in improved physical stability of the vesicles.

Synergistic Interaction between Cholesterol and Functionalized Ionic Liquid Based Surfactant Leading to the Morphological Transition

AbstractStable vesicular aggregate is the primary requisite to transport and deliver the biological and pharmaceutical standards. The serendipitous discovery of nonlipid vesicles made with the nonlipid building blocks, such as ionic liquid based surfactants (ILBSs) proved to be advantageous over others. In a quest to achieve stable, more rigid and hydrophobic vesicles from ILBSs, here we have investigated the lipid (cholesterol) induced unilamellar vesicle formation in the aqueous functionalized and nonfunctionalized ILBSs. The vesicles show outstanding stability with time, temperature and dilution with water. Spectroscopic (turbidity, steady state absorbance and fluorescence), scattering (dynamic light scattering) and microscopic (transmission electron microscopy) techniques were used to characterize the vesicles. The hydration behavior and rigidity associated with the vesicular bilayer on transforming from micellar assembly was characterized through steady state absorbance and fluorescence techniques. The unilamellar vesicles derived are compared with the conventional surfactant, i. e. cetyltrimethyl ammonium bromide.

Enhanced Physical Stability of Mixed Ion Pair Amphiphile/Double-chained Cationic Surfactant Vesicles in the Presence of Cholesterol

In this study, a pseudodouble-chained ion pair amphiphile (IPA), hexadecyltrimethylammoniumdodecylsulfate (HTMA-DS), and dialkyldimethylammonium bromide (DXDAB) with different chain lengths were used as the main materials to fabricate positively charged catanionic vesicles with various mole fractions of cholesterol. The effects of cholesterol and DXDAB alkyl chain length on physical stability of the catanionic vesicles were then investigated by size, zeta potential, and Fourier transform infrared analyses. With the presence of cholesterol in the mixed HTMA-DS/DXDAB vesicles or with increasing the DXDAB content in the presence of a proper amount of cholesterol, the physical stability of the catanionic vesicles could be enhanced. The spacing effect of cholesterol would reduce the counterion binding tendency at the charged vesicle surfaces, resulting in a more pronounced charge character of the catanionic vesicles. Furthermore, cholesterol-induced disordered structure contributed to the flexibility of the vesicular bilayers. Thus the physical stability of the vesicles was improved by adding cholesterol. With increasing the hydrocarbon chain length of DXDAB, cholesterol located toward the middle of the bilayers, enhancing the effects of cholesterol on charge and molecular packing characteristics of the vesicles. This led to a more pronounced stability enhancement effect on the vesicles with a longer alkyl chain length of DXDAB. The results suggested that the presence of cholesterol in the HTMA-DS/DXDAB catanionic vesicles could enhance vesicle stability through adjusting intra-vesicle and/or inter-vesicle interactions. In addition, the stability enhancement effect was more pronounced in the systems with a long DXDAB alkyl chain. The findings will be useful for developing new formulas of catanionic vesicles as drug delivery carriers.

Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer.

Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and 31P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.

Real-time analysis of protein and protein mixture interaction with lipid bilayers

Artificial lipid bilayers in the form of planar supported or vesicular bilayers are commonly used as models for studying interaction of biological membranes with different substances such as proteins and small molecule pharmaceutical compounds. Lipid membranes are typically regarded as inert and passive scaffolds for membrane proteins, but both non-specific and specific interactions between biomolecules and lipid membranes are indeed ubiquitous; dynamic exchange of proteins from the environment at the membrane interface can strongly influence the function of biological membranes. Such exchanges would either be of a superficial (peripheral) or integrative (penetrating) nature. In the context of viral membranes (termed envelopes), this could contribute to the emergence of zoonotic infections as well as change the virulence and/or pathogenicity of viral diseases. In this study, we analyze adsorption/desorption patterns upon challenging tethered liposomes and enveloped virus particles with proteins – or protein mixtures - such as bovine serum albumin, glycosylphosphatidylinositol anchored proteins and serum, chosen for their different lipid-interaction capabilities. We employed quartz crystal microbalance and dual polarization interferometry measurements to measure protein/membrane interaction in real time. We identified differences in mass uptake between the challenges, as well as differences between variants of lipid bilayers. Tethered viral particles showed a similar adsorption/desorption behavior to liposomes, underlining their value as model system. We believe that this methodology may be developed into a new approach in virology and membrane research by enabling the combination of biophysical and biochemical information.

Interactions between sodium dodecylsulphate and didodecyldimethylammonium bromides vesicles in aqueous solutions

The calorimetric results of heat of dilution of SDS aqueous solution [0.0348molkg−1] in water and DDAB vesicle solution [0.002molkg−1] were applied to estimate the enthalpies of interactions between an anionic surfactant SDS and vesicles formed by DDAB in aqueous solutions at 25°C and 40°C. They are negative in the whole investigated SDS concentration range at both temperatures. The enthalpy of interaction changes illustrates the difference between the SDS monomers energetic state in water and that in the DDAB vesicle bilayers. The plots of interaction enthalpy against SDS concentration have completely different shapes at both temperatures. These have been discussed in terms of electrostatic interactions between: positively charged lipid head groups with oppositely charged SDS molecules, which penetrate the vesicular bilayers. It appeared that dependence of interaction enthalpy on SDS concentration can be used to reveal the phase transitions from a liquid crystal to gel state in vesicular system bilayers. Adding SDS to DDAB vesicle solution affects significantly, electrostatic interactions of vesicular bilayers leading to changes of aggregates structural features that have been confirmed by transmission electron microscope examinations.

Interplay between alkyl chain asymmetry and cholesterol addition in the rigid ion pair amphiphile bilayer systems.

Ion pair amphiphile (IPA), a molecular complex composed of a pair of cationic and anionic surfactants, has been proposed as a novel phospholipid substitute. Controlling the physical stability of IPA vesicles is important for its application developments such as cosmetic and drug deliveries. To investigate the effects of IPA alkyl chain combinations and the cholesterol additive on the structural and mechanical properties of IPA vesicular bilayers, we conducted a series of molecular dynamics studies on the hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS) and dodecyltrimethylammonium-hexadecylsulfate (DTMA-HS) IPA bilayers with cholesterol. We found that both IPA bilayers are in the gel phase at 298 K, consistent with experimental observations. Compared with the HTMA-DS system, the DTMA-HS bilayer has more disordered alkyl chains in the hydrophobic region. When adding cholesterol, it induces alkyl chain ordering around its rigid sterol ring. Yet, cholesterol increases the molecular areas for all species and disturbs the molecular packing near the hydrophilic region and the bilayer core. Cholesterol also promotes the alkyl chain mismatch between the IPA moieties, especially for the DTMA-HS bilayer. The combined effects lead to non-monotonically enhancement of the membrane mechanical moduli for both IPA-cholesterol systems. Furthermore, cholesterol can form H-bonds with the alkylsulfate and thus enhance the contribution of alkylsulfate to the overall mechanical moduli. Combined results provide valuable molecular insights into the roles of each IPA component and the cholesterol on modulating the IPA bilayer properties.

Composite chitosan-transfersomal vesicles for improved transnasal permeation and bioavailability of verapamil

The creation of composite systems has become an emerging field in drug delivery. Chitosan has demonstrated several pharmaceutical advantages, especially in intranasal delivery. In this manuscript, a comparative study was conducted between regular vesicles (transfersomes and penetration enhancer vesicles) and composite vesicles (chitosan containing transfersomes and penetration enhancer vesicles) loaded with a model antihypertensive drug; verapamil hydrochloride VRP. Composite vesicles displayed larger particle size than regular vesicles owing to the coating potential of chitosan on the vesicular bilayer as displayed by transmission electron microscopy, with an increased viscosity of composite vesicles and a shift in the zeta potential values from negative to positive. The entrapment efficiency of VRP in the vesicles ranged from 24 to 64%, with best physical stability displayed with transfersomal vesicles prepared using sodium deoxycholate. Chitosan slowed the in vitro release of VRP from the selected formulation but managed to achieve high penetrability across sheep nasal mucosa as displayed by confocal laser microscopy. The chitosan composite transfersomal formulation exhibited absolute bioavailability of 81.83% compared to the oral solution which displayed only 13.04%. Findings of this manuscript highly recommend chitosan as a promising functional additive in vesicular formulations to improve the intranasal delivery of drugs with low oral bioavailability.

The formation and stability of sodium dodecylsulfate vesicles mediated by rough glass surface

Under the mediation of a rough glass surface (RGS), formation of vesicles was realized from a micellar solution of only a simple anionic single-tailed surfactant (STS), sodium dodecylsulfate (SDS), to form a mixed solution of micelles and vesicles. Interestingly, the SDS vesicles formed in the solution displayed good stability upon long-term storage, exposure to high temperature, freeze–thawing, and high salinity after the RGS removal. Adsorption and water contact angle measurements demonstrated that SDS could adsorb on the RGS to form bilayers in which the molecular packing parameter of SDS was in the range of 1/2–1. The bilayer adsorption and the roughness of the solid surface played an important role in vesicle formation. AFM observations suggested that the alkyl tails of SDS in the vesicular bilayer membranes were partially interdigitated, and molecular dynamics simulations revealed that the partially interdigitated bilayer structure is crucial for the formation and stability of the SDS vesicles. This work contributes to the understanding of the nature of vesicle systems.

Maximizing the Therapeutic Efficacy of Imatinib Mesylate–Loaded Niosomes on Human Colon Adenocarcinoma Using Box-Behnken Design

This research purposed to formulate an optimized imatinib mesylate (IM)–loaded niosomes to improve its chemotherapeutic efficacy. The influence of 3 formulation factors on niosomal vesicular size (Y1), zeta potential (Y2), entrapment capacity percentage (Y3), the percentage of initial drug release after 2 h (Y4), and the percentage of cumulative drug release after 24 h (Y5) were studied and optimized using Box-Behnken design. Optimum desirability was specified and the optimized formula was prepared, stability tested, morphologically examined, checked for vesicular bilayer formation and evaluated for its in vitro cytotoxicity on 3 different cancer cell lines namely MCF-7, HCT-116, and HepG-2 in addition to 1 normal cell line to ensure its selectivity against cancer cells. The actual responses of the optimized IM formulation were 425.36 nm, −62.4 mV, 82.96%, 18.93%, and 89.45% for Y1, Y2, Y3, Y4, and Y5, respectively. The optimized IM-loaded niosomes confirmed the spherical vesicular shape imaged by both light and electron microscopes and further proven by differential scanning calorimetry. Moreover, the optimized formula exhibited improved stability on storage at 4 ± 2°C and superior efficacy on MCF7, HCT-116, and HepG2 as IC50 values were 6.7, 16.4, and 7.3 folds less than those of free drug, respectively. Interestingly, IC50 of the optimized formula against normal cell line was ranged from 3 to 11 folds higher than in different cancer cells indicating a higher selectivity of the optimized formula to cancer cells. In conclusion, the incorporation of IM in niosomes enhanced its efficacy and selectivity toward cancer cells, presenting a promising tool to fight cancer using this approach.

Elucidating GPCR Functional Dependence on Plasma Membrane Composition using Giant Unilamellar Protein-Vesicles

Using an agarose hydration technique for protein incorporation into vesicular bilayers, we elucidate the effects of membrane composition and ordering on G Protein Coupled Receptors (GPRCs). We successfully incorporate GPCRs into model membranes in the form of giant unilamellar protein-vesicles (GUPs). Using this completely in vitro platform we observe that the functional rate of the human serotonin receptor, GPCR 5-HT1A, and the A2a Adenosine GPCR is dependent on membrane composition and ordering. We use BODIPY-GTPγS as our fluorescent marker to track the irreversible exchange between GDP and GTP on G proteins over time in GUPs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), brain sphingomyelin (BSM), and cholesterol (Chol) as well as synthetic lamellar phase diblock copolymer. Furthermore, using this approach we demonstrate that the incorporated receptors display a biased orientation with the N-terminus located on the exterior (extracellular) and the C-terminus on the interior (cytosolic).

Novel synthesis of biologically active CdS nanoclusters in cell-mimicking vesicles

ABSTRACTStable cadmium sulphide (CdS) nanoclusters have been synthesised in non-ionic surfactant vesicles (niosomes). The nanocluster characteristics show strong dependence on the Cd2+ precursor concentration. At low precursor concentration, aggregates comprising fine nanocrystals of CdS with diameter 2–5 nm are formed. At high precursor concentrations, crystalline CdS nanoclusters (15–20 nm in diameter) are formed. Interestingly, these nanoclusters reside exclusively in the vesicular bilayer. Moreover, these nanoclusters are highly photoluminescent and exhibit cytotoxicity towards cancerous HeLa cells. As the vesicular bilayer mimics the cell membrane, the present work is extremely relevant to the use of the CdS nanoclusters for diagnostic and therapeutic purpose.

Exploring physical stability characteristics of positively charged catanionic vesicle/DNA complexes

Cationic vesicles fabricated from various novel materials have been recently developed for DNA delivery applications, and the physical stability of the vesicle/DNA complexes played an important role in the transfection efficiency. In this study, positively charged catanionic vesicles composed of hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), ditetradecyldimethylammonium bromide (DTDAB), and cholesterol were prepared in buffer solution by a forced formation approach, and the association behavior of the catanionic vesicles with two types of DNA molecules with the consideration of the vesicle/DNA complex stability was explored by dynamic light scattering, fluorescence polarization, and DNA electrophoresis techniques. In a buffer solution of pH 7.4, HTMA-DS/DTDAB/cholesterol catanionic vesicles with high physical stability and positive charge character were successfully fabricated. A toxicity examination then implied that the catanionic vesicles have the potential of being applied in transfection applications. It is noted that the catanionic vesicle dispersions became unstable as prepared from a concentrated one by dilution due to the less charged vesicle surfaces and the more rigid vesicular bilayers, which could be explained by the enhanced dissolution of HTMA+ from the vesicles. The physical stability and charge character of the catanionic vesicle/DNA complexes were found strongly dependent on the mixing ratio of the vesicle to DNA. The formation of the catanionic vesicle/DNA complexes was also confirmed by an electrophoresis analysis. Moreover, not only the electrostatic repulsion between the vesicle/DNA complexes but also the molecular packing of the vesicular bilayers played roles in governing the physical stability of the vesicle/DNA complexes.

Direct Measurement of Dipole Electric Field in Model Membranes using Vibrtaional Shifts of P-Cyanophenylalanine and Coupled with Molecular Dynamics Simulations

The composition of biological lipid bilayer membrane creates a complex structural and electrostatic environment that regulates important membrane functions. The alignment of molecular dipole moments from lipid head groups and water molecules located at membrane-water interface creates the dipole potential (Vd). The dipole field (Fd) generated from this potential is the largest and about 1-10 MV/cm in magnitude, determined by indirect measurement techniques. It is located entirely within the membrane interior, and therefore, direct measurement and manipulation of Fd is a challenge. Here, we present direct measurement of dipole electrostatic field within vesicular bilayer using vibrational Stark effect (VSE) shifts of nitrile oscillator in response to the local electrostatic field. We intercalated four different α-helix transmembrane peptides containing unnatural amino acid, p-cyanophenylalanine (p-CN-Phe) at four unique positions into unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Molecular Dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid and one buried in the interior of the bilayer were performed to examine the structure of nitrile with respect to the membrane normal, the assumed direction of Fd, by quantifying both tilt of the helix in bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. As nitrile systematically moved towards the membrane interior, the vibrational absorption energies of nitrile showed blue shift. We used the measured VSE shifts and nitrile orientations within the membrane and calculated the magnitude of Fd to be 8 −11 MV/cm, within the range reported in the literature. We increased the chemical and structural complexity of model membrane by adding cholesterol into vesicle composition at different concentrations and determined that Fd changes as function of membrane composition and temperature.