Self‐assembly of pH and calcium dual‐responsive peptide‐amphiphilic hydrogel

Peptide‐based hydrogels have gained much interest for biomedical applications as a result of their biocompatibility. Herein, we reported a synthetic pH‐sensitive and calcium‐responsive peptide‐amphiphilic hydrogel. The sequences of the peptide amphiphiles were derived from the repeat‐in‐toxin (RTX) motif. At a certain peptide‐amphiphile concentration, self‐assembly was accompanied by the formation of a rigid, viscoelastic hydrogel at low pH or the presence of calcium ions. Circular dichroism spectra showed that the peptide amphiphiles adopted beta‐sheet structure. Meanwhile, as revealed by transmission electron microscopy, the peptide‐amphiphile self‐assembly was accompanied by the formation of long interconnected nanofibrillar superstructure. Material properties of the resulting peptide‐amphiphile hydrogel were characterized using oscillatory sheer rheology, and the storage modulus (G′) was found to be one order of magnitude higher than the loss modulus (G″), indicating a moderately rigid viscoelastic material. Furthermore, with systematical residue substitution, it was found that the aspartic acid within the repeat‐in‐toxin sequence of peptide amphiphiles was responsible for the pH and calcium selectivity. The environmental responsiveness, secondary structure, morphology, and mechanical nature of the peptide‐amphiphile hydrogel make it a possible material candidate for biomedical and engineering application. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Deciphering the role of charge,hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile acid based facial amphiphiles

We synthesized four cationic bile acid based facial amphiphiles featuring trimethyl ammonium head groups. We evaluated the role of these amphiphiles for cytotoxic activities against colon cancer cells and their membrane interactions by varying charge, hydration and hydrophobicity. The singly charged cationic Lithocholic acid based amphiphile (LCA-TMA₁) is most cytotoxic, whereas the triply charged cationic Cholic acid based amphiphile (CA-TMA₃) is least cytotoxic. Light microscopy and Annexin-FITC assay revealed that these facial amphiphiles caused late apoptosis. In addition, we studied the interactions of these amphiphiles with model membrane systems by Prodan-based hydration, DPH-based anisotropy, and differential scanning calorimetry. LCA-TMA₁ is most hydrophobic with a hard charge causing efficient dehydration and maximum perturbations of membranes thereby facilitating translocation and high cytotoxicity against colon cancer cells. In contrast, the highly hydrated and multiple charged CA-TMA₃ caused least membrane perturbations leading to low translocation and less cytotoxicity. As expected, Chenodeoxycholic acid and Deoxycholic acid based amphiphiles (CDCA-TMA₂, DCA-TMA₂) featuring two charged head groups showed intermediate behavior. Thus, we deciphered that charge, hydration, and hydrophobicity of these amphiphiles govern membrane interactions, translocation, and resulting cytoxicity against colon cancer cells.     

Hemocyanin of the chiton, Stenoplax conspicua (Dall)

1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 X 10(6) daltons, (dt) and a sedimentation coefficient of 60 S. 2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 10(5) and 4.17-4.75 x 10(5) dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly. 3. The pH dependence of the molecular weights from pH 4.5 to 11 exhibit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer. 4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins. 5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+, and analyzed by means of the decamer-dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (Napp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.     

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.     

Preparation of gemini-type amphiphiles bearing cyclitol head groups and their application as high-performance modifiers for lipases

Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N'-[iminobis(trimethylene)]bisquinamide, prepared from iminobispropylamine and quino-1,5-lactone, and dialkyl N-(3-carboxypropanoyl)-L-glutamates as polar and hydrophobic components, respectively. Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL) are co-lyophilized with these synthesized gemini-type amphiphiles, and their transesterification activities in organic solvents are evaluated. The modified PCL and CRL prepared by using each amphiphile showed highly enhanced and moderately enhanced enzyme activity, respectively. These results are discussed in terms of the increased preferential exclusion of the hydrophilic heads of the amphiphile and of the topological view of the amphiphile.     

Synthesis and structure of a vesicular assembly of dialkylammonium-type amphiphile carrying L-3-(3-N-ethylcarbazolyl)alanine

Summary A novel unnatural amino acid, L-3-(3-N-ethylcarbazolyl)alanine, was synthesized and used for synthesis of a chiral dialkylammonium-type amphiphile. The synthetic amphiphile formed a vesicular assembly in aqueous dispersion showing gel-liquid crystalline phase transition around 25 °C. The bilayer membrane composed of the amphiphile was nearly free from excimer as evidenced by fluorescence spectroscopy.     

Enhancement of Candida rugosa lipase activity in non-aqueous media by co-lyophilization with amphiphiles from aqueous solution

Modified Candida rugosa lipase was co-lyophilized with two gemini-type amphiphiles, l- and d-2-(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionylamino)-pentanedioic acid didodecyl ester or dodecanoic acid 2-[(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionyl)-(2-dodecanoyloxy-ethyl)-amino]-ethyl ester. Enzymatic activities of the modified lipases in the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in cyclohexane were enhanced as much as by 37-78, 1.5–5- and 41–83-fold of magnitude relative to that of native enzyme, respectively. The lack of significant enhancement of the enzymatic activity, only in the case of the d-isomeric amphiphile-modified lipase, was considered from the topological view of the amphiphile.     

New chiral fluoroanthryl derivatives: Resolution of the enantiomers by chromatography on cellulose esters and their evaluation as chiral solvating agents in NMR spectroscopy

2,2,2-Trifluoro-1-(9-anthryl)ethanol (TFAE) has now been widely used as a powerful chiral solvating agent for NMR spectroscopy. In connection with the development of a new general synthesis of halogenoalkylalkanols, starting from the corresponding ketone or aldehyde, we synthesized some halogenoalkyl-1-(9-anthryl)methanol derivatives liable to work as chiral solvating agents. The racemic anthryl derivatives were preparatively resolved into their corresponding enantiomers by chromatography on triacetyl cellulose (CTA I) or on meta-methylbenzoyl cellulose beads as chiral stationary phases. Their effectiveness as chiral solvating agents was measured as the magnitude of the splitting induced in the ¹H-NMR spectra of 1-phenylethylamine and of (1-phenylethyl)methyl ether in comparison with splitting caused by TFAE. While TFAE induced the largest splitting for 1-phenylethylamine, 2,2,3,3,3-pentafluoro-1-(9-anthryl)propanol 2 was more effective in the case of (1-phenylethyl)methyl ether, pointing out that depending on the substrate, other derivatives of the TFAE type can be very useful as chiral solvating agents.     

Characterization of the Streptococcus adjacens group antigen structure.

Serological classification of bacteria requires the presence of an antigen unique to the organism of interest. Streptococci are serologically differentiated by group antigens, many of which are carbohydrates, although some are amphiphiles. This report describes the chemical characterization of the Streptococcus adjacens group antigen structure. Previous studies demonstrated that the amphiphile contained phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids. Phosphodiester bonds present in the purified group antigen were identified as part of a poly(ribitol phosphate), since ribitol phosphate was the only organic phosphate detected after acid hydrolysis. Hydrofluoric acid cleavage of the phosphodiester bonds generated oligosaccharide repeating units. Gas chromatography-mass spectrometric analysis of the methylated, acetylated oligosaccharide suggested that the repeating unit is a trisaccharide of Galp beta 1-3Galp beta 1-4GalNac with N-acetylgalactosamine attached in beta-linkage to either the number two or the number four carbon of ribitol. The lipid- and carbohydrate-substituted poly(ribitol phosphate) of the S. adjacens group antigen therefore is a unique amphiphile structure, differing in its repeating-unit structure from the polyglycerophosphate structure of the more common gram-positive amphiphile lipoteichoic acid.     

Controlled disassembly of peptide amphiphile fibres.

In this paper, the introduction of both a methionine residue and a nitrobenzyl derivative as a labile linker between the peptide part and the hydrophobic alkyl chain of a peptide amphiphile are presented. These modifications are shown not to inhibit the formation of structured assemblies that analogous peptide amphiphiles lacking the linkers are able to form. Moreover, the introduction of either labile linker allows removal of the peptide amphiphile's stabilizing hydrophobic moieties to initiate a controlled disassembly of fibre aggregates. This is achieved by either treatment with CNBr or UV irradiation, respectively. These disassembly mechanisms could be the starting point for methodology that allows further manipulation of self-assembled peptide amphiphile architectures.     

Nutritionally variant streptococcal serotype I antigen. Characterization as a lipid-substituted poly(ribitol phosphate)

Initial characterization of the nutritionally variant streptococcal serotype I amphipathic polymer indicated an estimated m.w. of 360,000 for the aggregated form of the molecule, whereas the dissociated form had an estimated m.w. of 38,000 based on dextran m.w. standards. In addition, SDS-PAGE indicated an amphiphile with a stepladder appearance made up of several components with m.w. larger than the major electrophoresis components of LPS or lipoteichoic acid. Chemically, the serotype I amphiphile appeared to be a lipid-substituted poly(ribitol phosphate) with galactose and alanine substitution on the ribitol phosphate backbone. This report represents the first characterization of a bacterial amphiphile with a chemical composition and structure as proposed here. As is the case with other bacterial amphiphiles, the nutritionally variant streptococcal serotype I amphiphile was found both intra- and extracellularly. Finally, immunofluorescence studies demonstrated that the amphiphile was expressed on the cell surface.     

Amphiphile-induced phosphatidylserine exposure in human erythrocytes

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time -and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37°C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.     

Structural characterization of bacteriophage M13 solubilization by amphiphiles

The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

Amphiphiles for supercritical CO2

A semi-fluorinated hybrid amphiphile, pentadecafluoro-5-dodecyl (F7H4) sulfate, has been shown to form reversed micelles in dense CO₂; the aggregates evolve to form water-in-CO₂ (w/c) microemulsion droplets on addition of water. Aggregation structures in these w/c phases have been characterised by small-angle neutron scattering (SANS), showing the presence of cylindrical droplets, which change into dispersed lamellar phases at even higher water loadings. Other systems are also introduced, being high internal phase emulsions (HIPEs) with brine, and liquid and supercritical CO₂, stabilized by certain commercially available nonylphenol ethoxylates (Dow Tergitol NP-, and Huntsman Surfonic N- amphiphiles). These dispersions have been characterised by SANS for the first time. Quantitative analyses of the HIPEs SANS profiles show that they behave similarly to hydrocarbon-water emulsion analogues, with regard to total interfacial areas and the effects of amphiphile concentration on the underlying structures. Finally, the advantages and disadvantages of both approaches for controlling the physico-chemical properties of liquid/supercritical CO₂ in potential applications are compared and contrasted. These results highlight the importance of using specially designed CO₂-philic amphiphiles for generating self-assembly structures in dense CO₂.     

Dual modulation of protein kinase C activity by sphingosine.

Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N'-cholesteryl amido-ethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted with caution because of this dual role of the amphiphile.     

Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes

Novel cationic amphiphilic compounds were prepared from vernonia oil, a natural epoxidized triglyceride, and studied with respect to vesicle formation, encapsulation of biomaterials such as DNA, and their physical stability and transport through isolated plant cuticle membranes. The amphiphiles studied were a single-headed compound III (a quaternary ammonium head group with two alkyl chains) and a triple-headed compound IV, which is essentially three molecules of compound III bound together through a glycerol moiety. Vesicles of the two amphiphiles, prepared by sonication in water and solutions of uranyl acetate or the herbicide 2,4-D (2,4-dichloropenoxy acetic acid), were examined by TEM, SEM, AFM, and confocal laser systems and had a spherical shape which encapsulated the solutes with diameters between 40 and 110 nm. Vesicles from amphiphile IV could be made large enough to encapsulate a condensed 5.2kb DNA plasmid (pJD328). Vesicles of amphiphile IV were also shown to pass intact across isolated plant cuticle membranes and the rate of delivery of encapsulated radio-labeled 2,4-D through isolated plant cuticle membranes obtained with these vesicles was clearly greater in comparison to liposomes prepared from dipalmitopyl phosphatidylcholine (DPPC) and the control, nonencapsulated 2,4-D. Vesicles from amphiphiles III and IV were found to be more stable than those of liposomes from DPPC. The data indicate the potential of vesicles prepared from the novel amphiphile IV to be a relatively efficient nano-scale delivery system to transport DNA and other bioactive agents through plant biological barriers. This scientific approach may open the way for further development of efficient in vivo plant transformation systems.     

Chiral resolutions of (9-anthryl)methoxyacetic acid and (9-anthryl)hydroxyacetic acid by capillary electrophoresis

The resolutions of (9-anthryl)methoxyacetic acid (9AMAA) and (9-anthryl)hydroxyacetic acid (9AHAA) were performed by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as a chiral selector. Various factors affecting migration time and resolutions of these compounds were investigated with a run voltage of 20 kV, column temperature 20 degrees C and 20 mM Tris-H(3)PO(4) buffer (pH 6.5) containing 5 mM HP-beta-CD for 9AMAA, or 10 mM HP-beta-CD for 9AHAA, (+/-)-9AMAA and (+/-)-9AHAA were successfully separated at Rs 3.27 and 1.92, respectively.     

The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.     

Morphological characterization of exovesicles and endovesicles released from human erythrocytes following treatment with amphiphiles.

In order to morphologically characterize exo- and endovesicles released during treatment of erythrocytes with amphiphiles and to look for possible amphiphile-specific effects on the vesiculation pattern, human erythrocytes were treated at 37 degrees C with amphiphiles at concentrations where they exhibit maximum protection against hypotonic haemolysis (cAHmax). Released exo-and endovesicles and treated cells were studied by means of transmission (TEM) and scanning (SEM) electron microscopy. All sphero-echinocytogenic amphiphiles induced a release of both spherical and tubular exovesicles. Dodecyl maltoside, a nonionic amphiphile with a bulky polar head, induced a release of predominantly tubular exovesicles, while all other sphero-echinocytogenic amphiphiles induced a release of predominantly spherical exovesicles. Some branched tubular exovesicles were released by a double-chained cationic amphiphile. Tail- and tongue-like structures were often seen on the exovesicles. Spherical exovesicles were frequently invaginated. Stomatocytogenic amphiphiles induced endovesiculation. In erythrocytes treated with most of the stomatocytogenic amphiphiles the endovesicles were clustered, but with some amphiphiles the endovesicles were randomly distributed. Large ringformed endovesicles (octaethyleneglycol alkyl ethers) and endovesicles in chains (octyl and decyl glucopyranoside) also occurred. The endovesicle membrane was often budding onto the lumen of the vesicle and in some cases this could ultimately lead to a vesicle inside the endovesicle. We conclude that amphiphiles do not only trigger vesiculation, but may also specifically affect the vesiculation processes.