Cationic Vesicles Research Articles

Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles

Bolaamphiphilic cationic vesicles with acetylcholine (ACh) surface groups were investigated for their ability to deliver a model protein–bovine serum albumin conjugated to fluorescein isothiocyanate (BSA-FITC) across biological barriers in vitro and in vivo. BSA-FITC-loaded vesicles were internalized into cells in culture, including brain endothelial b.End3 cells, at 37°C, but not at 4°C, indicating an active uptake process. To examine if BSA-FITC-loaded vesicles were stable enough for in vivo delivery, we tested vesicle stability in whole serum. The half-life of cationic BSA-FITC-loaded vesicles with ACh surface groups that are hydrolyzed by choline esterase (ChE) was about 2h, whereas the half-life of vesicles with similar surface groups, but which are not hydrolyzed by choline esterase (ChE), was over 5h. Pyridostigmine, a choline esterase inhibitor that does not penetrate the blood-brain barrier (BBB), increased the stability of the ChE-sensitive vesicles to 6h but did not affect the stability of vesicles with ACh surface groups that are not hydrolyzed by ChE. Following intravenous administration to pyridostigmine-pretreated mice, BSA-FITC encapsulated in ChE-sensitive vesicles was distributed into various tissues with marked accumulation in the brain, whereas non-encapsulated (free) BSA-FITC was detected only in peripheral tissues, but not in the brain. These results show that cationic bolaamphiphilic vesicles with ACh head groups are capable of delivering proteins across biological barriers, such as the cell membrane and the blood-brain barrier (BBB). Brain ChE activity destabilizes the vesicles and releases the encapsulated protein, enabling its accumulation in the brain.

Direct formation of cationic polypeptide vesicle as potential carrier for drug and gene

The pH-responsive polypeptide grafted with polycation was prepared through copper(I)-catalyzed “click chemistry”. The amphiphilic polypeptide directly formed into cationic vesicle when it was dissolved in phosphate buffer solution (PBS). The hydrophilic DOX·HCl was loaded into the hollow core of vesicle. The in vitro release behavior of DOX·HCl from vesicle in PBS could be adjusted by the pH of release media. In vitro cell experiments demonstrated that the DOX·HCl loaded vesicle showed effective cellular proliferation inhibition. In addition, the preliminary gel retardation assay revealed that PLG-g-PAMA could efficiently bind to DNA, indicating its potential use as gene carrier. The more in-depth studies of PLG-g-PAMA vesicle for drug and gene co-delivery are in progress.

Catanionic vesicles from an amphiphilic prodrug molecule: a new concept for drug delivery systems

Toxicity and low entrapment efficiency are the main problems for pharmaceutical applications of catanionic vesicles. In order to minimize surfactant toxicity, increase the drug loading content and simultaneously reduce the need for tedious chemical synthesis, we use oleic acid as the biocompatible surfactant, which reacts with the selected drug molecules (amlodipine) to produce an amphiphilic prodrug molecule for the straightforward fabrication of catanionic vesicles. The prodrug molecules are easily obtained by proton transfer between amlodipine and oleic acid molecules. The characterization of prodrug molecules and their aggregation behaviours in aqueous solutions are investigated by using Fourier transform infrared spectrophotometry (FTIR), 1H-nuclear magnetic resonance (1H-NMR), differential scanning calorimetry (DSC), surface tension measurement, transmission electron microscopy (TEM), dynamic light scattering (DLS), conductivity and zeta potential (ζ). The results demonstrate that vesicles could be easily formed with the prodrug amphiphilic molecules dispersed in aqueous solutions. Particularly, the drug release behaviour of the as-prepared catanionic vesicles exhibits excellent sustained drug release properties, which demonstrates their promising application in the newly designed drug delivery system.

Diffusion of organic dyes in a niosome immobilized on a glass surface using fluorescence correlation spectroscopy

Giant multilameller niosomes containing cholesterol and triton X-100 are studied using fluorescence correlation spectroscopy (FCS). Dynamic light scattering (DLS) data indicates formation of niosomes of broadly two different sizes (diameter)--~150 nm and ~1300 nm. This is confirmed by field emission scanning electron microscopy (FE-SEM) and confocal microscopy. The diffusion coefficient (D(t)) of three organic dyes in the niosome immobilized on a glass surface is studied using fluorescence correlation spectroscopy. On addition of the room temperature ionic liquids (RTIL) (1-methyl-3-pentylimidazolium bromide, [pmim][Br] and 1-methyl- 3-pentylimidazolium tetra-fluoroborate, [pmim][BF(4)]) the size of the niosome particles increases. The D(t) of all the organic dyes (DCM, C343 and C480) increases on addition of RTILs, indicating faster diffusion. The viscosity calculated from the D(t) of the three dyes exhibits weak probe dependence. Unlike lipid or catanionic vesicle, the D(t) values in a niosome exhibit very narrow distribution. This indicates that the niosomes are fairly homogeneous with small variation of viscosity.

Interaction between GUVs and catanionic nanocontainers: new insight into spontaneous membrane fusion

Spontaneous receptor-free membrane fusion with pure lipid systems, used as a cell membrane model, is demonstrated with easy-to-handle lactose-derived catanionic vesicles. This fusion, mediated and controlled by phospholipids, emphasizes the great value of these nanovesicles for enhanced direct cytosolic drug delivery without the shortcomings linked with endocytic pathways.

Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles

The self-assembly of cationic and anionic amphiphile mixtures into vesicles in aqueous media was studied using two different systems: (i) decanoic acid and trimethyldecylammonium bromide and (ii) hexadecanedioic acid (a simple bola-amphiphile) and trimethyldecylammonium bromide. The resulting vesicles with varying amphiphile ratios were characterized using parameters such as the critical vesicle concentration, pH sensitivity, and encapsulation efficiency. We also produced and observed giant vesicles from these mixtures using the electroformation method and confocal microscopy. The mixed catanionic vesicles were shown to be more stable than those formed by pure fatty acids. Those containing bola-amphiphile even showed the encapsulation of a small hydrophilic solute (8-hydroxypyrene-1,3,6-trisulfonic-acid), suggesting a denser packing of the amphiphiles. Compression and kinetics analysis of monolayers composed of these amphiphiles mixtures at the air/water interface suggests that the stabilization of the structures can be attributed to two main interactions between headgroups, predominantly the formation of hydrogen bonds between protonated and deprotonated acids and the additional electrostatic interactions between ammonium and acid headgroups.

Bioavailability enhancement of zaleplon via proliposomes: Role of surface charge

The present systematic study focused to investigate the combined advantage of proliposomes and surface charge for improved oral delivery of zaleplon. The zaleplon loaded proliposomes were prepared using hydrogenated soyphosphatidylcholine (HSPC) and cholesterol (CHOL) in varying ratios, and the optimized formulation was tailored with dicetyl phosphate and stearylamine to obtain negative and positive charged vesicles, respectively. The formulations were characterized for micromeritics, size, zeta potential, and entrapment efficiency. Further, in vitro release and dissolution study carried out provide an insight on the stability and enhanced dissolution of zaleplon from proliposome formulations. The solid state characterization (SEM, DSC, and PXRD) studies unravel the transformation of zaleplon to amorphous or molecular state from the native crystalline form. To depict the conclusions, in situ single-pass perfusion and bioavailability studies were carried out in rats. The significant increase in effective permeability coefficient (Peff) and rate and extent of absorption from cationic vesicles indicate the importance of surface charge for effective uptake across the gastrointestinal tract. Overall a two- to fivefold enhancement in bioavailability in comparison with control confers the potential of proliposomes as suitable carriers for improved oral delivery of zaleplon.

Characterization and Aggregation Behaviors of Mixed DDAB/SDS Solution With and Without Poly(4-styrenesulfonic Acid-Co-Maleic Acid) Sodium

Synthetic vesicles are formed by cationic and anionic surfactants, didodecyldimethylammonium bromide (DDAB), and sodium dodecylsulfate (SDS). The morphology, size, and aqueous properties of cationic/anionic mixtures are investigated at various molar ratios between cationic and anionic surfactants. The charged vesicular dispersions made of DDAB/SDS are contacted or mixed with negatively charged polyelectrolyte, poly(4-styrenesulfonic acid-co-maleic acid) sodium (PSSAMA), to form complexes. Depending on DDAB/SDS molar ratio or PSSAMA/vesicle charge ratio, complexes flocculation or precipitation occur. Characterization of the cationic/anionic vesicles or complexes formed by the catanionic vesicles and polyelectrolytes is performed by transmission electron microscope (TEM), dynamic light scattering (DLS), conductivity, turbidity, and zeta potential measurements. The size, stability, and the surface charge on the mixed cationic/anionic vesicles or complexes are determined.

Gel formulations containing catanionic vesicles composed of alprenolol and SDS: Effects of drug release and skin penetration on aggregate structure

To fully utilize the extended contact time of gel formulations a novel formulation with drug containing catanionic aggregates offering prolonged drug release and skin penetration were investigated. This study aimed to further explore the drug release process from catanionic vesicles in gels.Catanionic vesicles were formed from alprenolol and sodium dodecyl sulphate. Physical gels composed of catanionic vesicles and a SoftCAT polymer were used as well as covalent Carbopol gels. Drug release was measured in vitro using a modified USP paddle method and the skin penetration was studied using dermatomized pig ear skin mounted in horizontal Ussing chambers. The aggregate structure was visualized with cryo-TEM during the drug release and skin penetration process.The study results show that catanionic vesicles are present in the formulations throughout the drug release process and during the clinically relevant skin application time. Hence, the decreased skin penetration rate stems from the prolonged release of drug substance from the gels. The rheological investigation shows that the gel structure of the physically cross-linked gels is maintained even as the drug substance is released and the gel volume is decreased. These findings indicate that the applicability of formulations like these is a future possibility.

Femtosecond study of ultrafast fluorescence resonance energy transfer in a catanionic vesicle

Ultrafast fluorescence resonance energy transfer (FRET) in a catanionic [sodium dodecyl sulfate (SDS)-dodecyltrimethyl ammonium bromide (DTAB)] vesicle is studied by femtosecond up-conversion. The vesicles (diameter ∼400 nm for SDS-rich and ∼250 nm for DTAB-rich vesicles) are much larger than the SDS and DTAB micelles (diameter ∼4 nm). In both micelle and vesicles, FRET occurs in multiple time scales and the time scales of FRET correspond to a donor-acceptor distance varying between 12 and 36 Å.

Mesoporous silica SBA-15 sheet with perpendicular nanochannels

Free-standing thin sheet form of mesoporous silica materials with perpendicular orientation is a much desired materials for its possible applications in catalysis, mask, and separation. A three component amphiphile system of sodium dodecyl sulfate/hexadecyltrimethylammonium bromide/pluronic-123(C16TMAB/SDS/P123) was employed to template the condensation of sodium silicates for the formation of SBA(⊥), a thin sheet of SBA-15 with perpendicular nanochannels. SBA(⊥) can be synthesized at SDS/C16TMAB=1.5 and T⩾40°C and shows pH-dependent morphology. It has uniform pore size ∼9nm, homogeneous sheet thickness in the range of 60–300nm and dimension of several microns. We studied in details the structure and morphology of the SBA(⊥) with variation of three experimental parameters: the SDS/C16TMAB ratio, the temperature, and the pH condition in the synthetic gel. It is proposed that the mixed surfactants of SDS and C16TMAB form catanionic vesicle in which the P123 and silicates are condensed. The balanced interaction of P123/silicate with the narrow confinement under surfactant bi-layers of C16TMAB/SDS promoted the formation of perpendicular nanochannels. Low temperature and pH conditions favor stronger segregation of the PPO and PEO–oligosilicate segments in the SBA(⊥) structure which gives the basis of thickness control of the sheet. The control of structure and morphology are discussed with modern theory of microphase separation in block copolymers under confinement.

Influence of Chain Length of Alcohols on Stokes’ Shift Dynamics in Catanionic Vesicles

In this paper, we explore the effects of the chain length of simple monohydroxy alcohol (C(n)OH, 2 ≤ n ≤ 8) and benzyl alcohol (C(6)H(5)CH(2)OH) upon the fluorescence dynamics of a dipolar solute probe, coumarin 153 (C153), in vesicles formed in aqueous solutions of two oppositely charged (cationic and anionic) surfactants in the presence of 0.05 mol kg(-1) alcohol. The catanionic vesicles are composed of 70 mol % sodium dodecyl sulfate (SDS) and 30 mol % cetyltrimethylammonium bromide (CTAB). The presence of alcohols of different chain length improves the stability of the catanionic vesicles. Dynamic light scattering (DLS) experiments suggest a gentle increase in the hydrodynamic diameter of the catanionic vesicle with alcohol chain length up to n=4 and then an abrupt increase for the rest of the alcohols considered. The polarity and dynamics of the catanionic vesicles, probed by the steady-state and time-resolved fluorescence spectroscopy, indicate a signature of confined water. Quantities measured from fluorescence experiments of these vesicles also show a mild variation for alcohols of chain length n ≤ 4, followed by a sudden variation for alcohols with n > 4. Interestingly, pentanol and benzyl alcohol in catanionic vesicles showed roughly similar effects due to their equivalent chain length. All of these data are remarkably correlated with the recently observed depression of the solubility temperature of catanionics with alcohol chain length (Langmuir2009, 25, 12516-12521).

N‐(Di)icosyl‐Substituted Benzo[a]phenoxazinium Chlorides: Synthesis and Evaluation as Near‐Infrared Membrane Probes

AbstractFive benzo[a]phenoxazinium chlorides containing alkyl chains with twenty carbon atoms at the 5‐ or 9‐positions of the tetracyclic ring were efficiently synthesised and characterised by UV/Vis and NIR spectroscopy. The absorption and emission maxima in ethanol occur in the range 627–641 nm and 645–676 nm, respectively, with quantum yields varying from 0.14 to 0.38. Preliminary photophysical studies with these fluorochromophores in zwitterionic [2,3‐bis(palmitoyloxy)propyl‐2‐(trimethylammonio)ethyl phosphate, DPPC] and cationic (N,N‐dimethyl‐N‐octadecyloctadecan‐1‐aminium bromide, DODAB) vesicles were carried out. The results showed that the new benzo[a]phenoxazinium derivatives are able to detect the gel to liquid‐crystalline lipid phase transition through variations in either the extent of H‐aggregation or in the acid–base equilibrium.

Simulation Study on the Properties of Cationic Lipid Bilayers and Vesicles

We are using molecular dynamics simulations to investigate the properties of ionizable cationic lipids forming bilayers and vesicles. This is important for rational design of lipid nanoparticle for delivery of nucleic acids (DNA, RNA). The process of transfection includes encapsulation of anionic biomolecules, binding of the nanoparticle to the membrane surface, endocytosis and release of the nucleic acids from the endosome. For ionizable cationic lipids the steps of this process are pH-dependent. In the acidic environment of the endosome, the charge density of the nanoparticle increases. Membrane disruption is believed to occur due to formation of the ion pairs between cationic lipids in the nanoparticle and anionic lipids in the endosome [Semple et al., Nature Biotech, 2010]. Due to favorable interactions between the headgroups, lipids in the ion pairs become cone-shaped and prone to form non-lamellar phases, such as the inverted hexagonal phase. We simulate cationic and anionic lipid mixtures in bilayers and vesicles and study the mechanism of membrane breakdown upon change of pH/charge density and variation in headgroup size.

Investigation on the aggregation behaviors of DDAB/NaDEHP catanionic vesicles in the absence and presence of a negatively charged polyelectrolyte

The aggregation behaviors of the cationic and anionic (catanionic) surfactant vesicles formed by didodecyldimethylammonium bromide (DDAB)/sodium bis(2-ethylhexyl) phosphate (NaDEHP) in the absence and presence of a negatively charged polyelectrolyte are investigated. The amount of the charge on the vesicle can be tuned by controlling the DDAB/NaDEHP surfactant molar ratio. The charged vesicular dispersions made of DDAB/NaDEHP are mixed with a negatively charged polyelectrolyte, poly(4-styrenesulfonic acid-co-maleic acid) sodium (PSSAMA), to form complexes. Depending on the polyelectrolyte/vesicle charge ratio, complex flocculation or precipitation occurs. Characterization of the catanionic vesicles and the complexes are performed by transmission electron microscope (TEM), Cryo-TEM, dynamic light scattering (DLS), conductivity, turbidity, zeta potential, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS) measurements.

Label-free spectroscopic detection of vesicles in water using vibrational sum frequency scattering

Vibrational sum frequency scattering (SFS) has been used to study sub-micron, catanionic vesicles in solution. The vesicles were synthesized from a binary mixture of dodecyltrimethylammonium bromide (DTAB) and sodium dodecylsulfate (SDS) surfactants in deuterated water, which spontaneously assemble into thermodynamically stable vesicles. The stability of these vesicles is attributed to a surfactant concentration asymmetry between the inner and outer bilayer leaflets. This concentration asymmetry should be observable by SFS due to local inversion symmetry-breaking. The signal corresponding to the symmetric sulfate stretch mode of the SDS head group is observed at 1042 cm−1, indicating that there is indeed asymmetry in the local structure of the leaflets. The results indicate that it should be possible to measure the interfacial structure of liposomes in aqueous solution and study in situ processes like the binding of sugars and proteins that are important for many processes in biophysical chemistry.

Vesicle-templated pH-responsive polymeric nanocapsules

We report the synthesis of pH responsive polymeric nanocapsules by templating unilamellar vesicles of dimethyldioctadecylammonium bromide (DODAB) using a RAFT-based templating approach. A short-chain living anionic copolymer containing randomly distributed acrylic acid and butyl acrylate units was first synthesized by RAFT in solution using dibenzyl trithiocarbonate (DBTTC) as the RAFT agent. The anionic copolymer chains were subsequently adsorbed onto the surface of cationic DODAB vesicles and were further chain extended to form a thick polymeric shell by feeding a monomer mixture comprising methyl methacrylate (MMA) and tertiary butyl acrylate (t-BA) in combination with the divinyl crosslinker ethylene glycol dimethacrylate (EGDMA) under starved feed conditions. CryoTEM characterization demonstrated successful formation of a thick crosslinked polymeric shell around the vesicles. Subsequent acid hydrolysis of the tertiary butyl ester groups of the crosslinked polymeric shell resulted in the formation of pH-responsive nanocapsules.

On the origin of the stability of foams made from catanionic surfactant mixtures

Using mixtures of the anionic myristic acid (C13COOH) and the cationic cetyl trimethylammonium chloride (C16TA+Cl−) in aqueous solutions at a 2:1 ratio, we show that the outstanding stability of foams generated from sufficiently concentrated “catanionic” surfactant mixtures can be explained by a synergy effect between two fundamentally different mechanisms. Applying a multi-scale approach, in which we link static and dynamic properties of the bulk solutions, isolated gas/liquid interfaces, thin liquid films and foams, we identify these two mechanisms to be as follows: firstly, cationic mixtures create tightly packed surfactant layers at gas/liquid interfaces, which are strongly viscoelastic and also confer high disjoining pressures when two interfaces are approaching each other to form a thin liquid film. Foams created with such kind of interfaces tend to be extremely stable against coalescence (film rupture) and coarsening (gas exchange). However, typical time scales to cover the interfaces are much longer than typical foaming times. This is why a second mechanism plays a key role, which is due to the presence of micron-sized catanionic vesicles in the foaming solution. The bilayers of these vesicles are in a gel-like state, therefore leading to nearly indestructible objects which act like elastic micro-spheres. At sufficiently high concentrations, these vesicles jam in the presence of the confinement between bubbles, slowing down the drainage of liquid during the initial foaming process and therefore providing time for the interfaces to be covered. Furthermore, the tightly packed vesicles strongly reduce bubble coalescence and gas transfer between bubbles.

Osmotically induced deformation of capsid-like icosahedral vesicles

We report the osmotic deformation of micron-size catanionic vesicles with icosahedral symmetry (20 faces, 12 vertices) upon incubation in small solutes (NaCl, glucose). The vesicles remain icosahedral at low osmotic pressure gradients across the bilayer, or spherical for outwards gradients. Above a threshold value of inwards pressure, the icosahedra develop a buckling instability: a depression is initiated at one or two ridges, grows until one vertex snaps into the icosahedra, leading to full collapse of one half of the vesicle into the other. Despite large local inversions in curvature, no release of encapsulated solutes is observed before the residual volume reaches negligible small values. Thin shell models correctly capture the buckling patterns of icosahedra in the low deformation limit. Comparison of experimental results with Monte Carlo simulations provides a first estimate for the conditions of shell disruption, and suggests it is predominantly driven by curvature rather than two-dimensional stretching or compression. The relevance of these results for the mechanics of viral capsids and controlled release applications is discussed.

Lipid chain geometry of C14 glycerol-based lipids: effect on lipoplex structure and transfection

The effects have been determined of a systematic alteration of the alkyl chain geometry of a C14 analogue of DOTMA on the detailed molecular architecture of the resulting cationic vesicles formed both in the absence and presence of 50 mol% DOPE, and of the lipoplexes prepared from these vesicles using either calf thymus or plasmid DNA. The C14 DOTMA analogues studied involved cis- or trans-double bonds at positions Δ9 or Δ11, and a compound (ALK) featuring an alkyne at position C9. For all of these analogues, examination by light scattering and neutron scattering, zeta potential measurement, and negative staining electron microscopy showed that there were no significant differences in the structures or charges of the vesicles or of the resulting lipoplexes, regardless of the nature of the DNA incorporated. Differences were observed, however, between the complexes formed by the various lipids when examining the extent of complexation and release by gel electrophoresis, where the E-lipids appeared to complex the DNA more efficiently than all other lipids tested. Moreover, the lipoplexes prepared from the E-lipids were the most effective in transfection of MDA-MB-231 breast cancer cells. As indicated through confocal microscopy studies, the E-lipids also showed a higher internalisation capacity and a more diffuse cellular distribution, possibly indicating a greater degree of endosomal escape and/or nuclear import. These observations suggest that the extent of complexation is the most important factor in determining the transfection efficiency of the complexes tested. At present it is unclear why the E-lipids were more effective at complexing DNA, although it is thought that the effective area per molecule occupied by the cationic lipid and DOPE head groups, and therefore the density of positive charges on the surface of the bilayer most closely matches the negative charge density of the DNA molecule. From a consideration of the geometry of the cationic lipids it is anticipated that the head groups of the E-lipids would occupy a smaller area per molecule than the ALK or Z-lipids.