Aggregation Behavior of Structurally Similar Therapeutic Peptides Investigated by 1H NMR and All-Atom Molecular Dynamics Simulations.

Understanding of peptide aggregation propensity is an important aspect in pharmaceutical development of peptide drugs. In this work, methodologies based on all-atom molecular dynamics (AA-MD) simulations and 1H NMR (in neat H2O) were evaluated as tools for identification and investigation of peptide aggregation. A series of structurally similar, pharmaceutically relevant peptides with known differences in aggregation behavior (D-Phe6-GnRH, ozarelix, cetrorelix, and degarelix) were investigated. The 1H NMR methodology was used to systematically investigate variations in aggregation with peptide concentration and time. Results show that 1H NMR can be used to detect the presence of coexisting classes of aggregates and the inclusion or exclusion of counterions in peptide aggregates. Interestingly, results suggest that the acetate counterions are included in aggregates of ozarelix and cetrorelix but not in aggregates of degarelix. The peptides investigated in AA-MD simulations (D-Phe6-GnRH, ozarelix, and cetrorelix) showed the same rank order of aggregation propensity as in the NMR experiments. The AA-MD simulations also provided molecular-level insights into aggregation dynamics, aggregation pathways, and the influence of different structural elements on peptide aggregation propensity and intermolecular interactions within the aggregates. Taken together, the findings from this study illustrate that 1H NMR and AA-MD simulations can be useful, complementary tools in early evaluation of aggregation propensity and formulation development for peptide drugs.

Physiological neutral pH drives a gradual lamellar-to-reverse cubic-to-reverse hexagonal phase transition in phytantriol-based nanoparticles.

Dispersed systems of bicontinuous cubic phases, called cubosomes, show a drug release rate faster than those obtained using other liquid-crystalline phases. To minimize side effects associated with the accelerated release of incorporated drugs, compounds may be added in the dispersions to produce systems of slow initial release and then fast release only in the desired action region. This paper addresses the addition of 10.0% (w/w) of decyl betainate chloride (DBC), a cleavable surfactant, into phytantriol/Pluronic-based dispersions to generate lamellar-to-cubic-to-hexagonal phase transitions. Small-angle X-ray scattering (SAXS) was used to analyze the mesophases obtained with the addition of DBC and pH variation. Transmission electron microscopy (TEM) images confirmed the presence of niosomes after the addition of DBC. The niosomes formed in these systems are pH-responsive with lamellar-to-hexosomes transitions at pH ≥ 7.4. The system investigated herein is gastro-resistant presenting potential therapeutic role for controlled release of drugs in neutral or alkaline environments of the organism.

Liquid crystal phantom for validation of microscopic diffusion anisotropy measurements on clinical MRI systems.

PURPOSE: To develop a phantom for validating MRI pulse sequences and data processing methods to quantify microscopic diffusion anisotropy in the human brain. METHODS: Using a liquid crystal consisting of water, detergent, and hydrocarbon, we designed a 0.5-L spherical phantom showing the theoretically highest possible degree of microscopic anisotropy. Data were acquired on the Connectome scanner using echo-planar imaging signal readout and diffusion encoding with axisymmetric b-tensors of varying magnitude, anisotropy, and orientation. The mean diffusivity, fractional anisotropy (FA), and microscopic FA (µFA) parameters were estimated. RESULTS: The phantom was observed to have values of mean diffusivity similar to brain tissue, and relaxation times compatible with echo-planar imaging echo times on the order of 100 ms. The estimated values of µFA were at the theoretical maximum of 1.0, whereas the values of FA spanned the interval from 0.0 to 0.8 as a result of varying orientational order of the anisotropic domains within each voxel. CONCLUSIONS: The proposed phantom can be manufactured by mixing three widely available chemicals in volumes comparable to a human head. The acquired data are in excellent agreement with theoretical predictions, showing that the phantom is ideal for validating methods for measuring microscopic diffusion anisotropy on clinical MRI systems. Magn Reson Med 79:1817-1828, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Interactions of PAMAM dendrimers with negatively charged model biomembranes.

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Soluble aggregates in aqueous solutions of polyion-surfactant ion complex salts and a nonionic surfactant.

Water-soluble aggregates based on two polyion-surfactant ion "complex salts", consisting of hexadecyltrimethylammonium (C16TA(+)) and polyacrylate (PA(-)) with either 25 or 6000 repeating units, with added nonionic surfactant octaethylene glycol monododecyl ether (C12E8) have been investigated. A previous phase study has shown that added C12E5 or C12E8 can solubilize complex salts in aqueous systems, and that increasing the poly(ethylene oxide) chain length of the nonionic surfactant and/or decreasing the polyion length favors dissolution. In this work we report on dynamic light scattering, NMR diffusometry, small-angle X-ray scattering, and isothermal titration calorimetry measurements performed to characterize the solubilized composite aggregates in dilute aqueous solution in terms of size and stoichiometry. It was found that mixed aggregates of polyacrylate, C16TA(+) ions, and C12E8, with almost constant stoichiometry, coexist with free micelles of C12E8 at all investigated mixing ratios. The length of the polyion only weakly affects the stoichiometry of the mixed aggregates while strongly affecting their size and water solubility.

Encapsulation of DNA in macroscopic and nanosized calcium alginate gel particles.

Calcium alginate beads, which are biodegradable and biocompatible, have been widely employed as delivery matrices for biomacromolecules. In the present work, the feasibility of encapsulation of DNA (which is used as a model biomacromolecule) in calcium alginate nanobeads (sub-200 nm size), prepared using a recently developed protocol based on the phase inversion temperature (PIT) emulsification method [Machado et al. Langmuir 2012, 28, 4131-4141], was assessed. The properties of the nanobeads were compared to those of the corresponding macroscopic (millimeter sized) calcium alginate beads. It was found that DNA, representing a relatively stiff and highly charged polyanion (thus like-charged to alginate), could be efficiently encapsulated in both nanosized and macroscopic beads, with encapsulation yields in the range of 77-99%. Complete release of DNA from the beads could be accomplished on dissolution of the gel by addition of a calcium-chelating agent. Importantly, the DNA was not denatured or fragmented during the preparation and collection of the nanobeads, which are good indicators of the mildness of the preparation protocol used. The calcium alginate nanobeads prepared by the herein utilized protocol thus show good potential to be used as carriers of sensitive biomacromolecules.

Supramolecular organization in self-assembly of chromatin and cationic lipid bilayers is controlled by membrane charge density.

In this work we have investigated the structures of aggregates formed in model systems of dilute aqueous mixtures of "model chromatin" consisting of either recombinant nucleosome core particles (NCPs) or nucleosome arrays consisting of 12 NCPs connected with 30 bp linker DNA, and liposomes made from different mixtures of cationic and zwitterionic lipids, 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The aggregates formed were characterized using different optical microscopy methods and small-angle X-ray scattering (SAXS), and the results are discussed in terms of the competing intermolecular interactions among the components. For a majority of the samples, the presence of lamellar structures could be identified. In samples with high fractions of DOTAP in the liposomes, well-defined lamellar structures very similar to those formed by the corresponding lipid mixtures and DNA alone (i.e., without histone proteins) were observed; in these aggregates, the histones are expelled from the model chromatin. The findings suggest that, with liposomes containing large fractions of cationic lipid, the dominating driving force for aggregation is the increase in translational entropy from the release of counterions, whereas with lower fractions of the cationic lipid, the entropy of mixing of the lipids within the bilayers results in a decreased DNA-lipid attraction.

Condensation and decondensation of DNA by cationic surfactant, spermine, or cationic surfactant-cyclodextrin mixtures: macroscopic phase behavior, aggregate properties, and dissolution mechanisms.

The macroscopic phase behavior and other physicochemical properties of dilute aqueous mixtures of DNA and the cationic surfactant hexadecyltrimethylammounium bromide (CTAB), DNA and the polyamine spermine, or DNA, CTAB, and (2-hydroxypropyl)-ß-cyclodextrin (2HPßCD) were investigated. When DNA is mixed with CTAB we found, with increasing surfactant concentration, (1) free DNA coexisting with surfactant unimers, (2) free DNA coexisting with aggregates of condensed DNA and CTAB, (3) a miscibility gap where macroscopic phase separation is observed, and (4) positively overcharged aggregates of condensed DNA and CTAB. The presence of a clear solution beyond the miscibility gap cannot be ascribed to self-screening by the charges from the DNA and/or the surfactant; instead, hydrophobic interactions among the surfactants are instrumental for the observed behavior. It is difficult to judge whether the overcharged mixed aggregates represent an equilibrium situation or not. If the excess surfactant was not initially present, but added to a preformed precipitate, redissolution was, in consistency with previous reports, not observed; thus, kinetic effects have major influence on the behavior. Mixtures of DNA and spermine also displayed a miscibility gap; however, positively overcharged aggregates were not identified, and redissolution with excess spermine can be explained by electrostatics. When 2HPßCD was added to a DNA-CTAB precipitate, redissolution was observed, and when it was added to the overcharged aggregates, the behavior was essentially a reversal of that of the DNA-CTAB system. This is attributed to an effectively quantitative formation of 1:1 2HPßCD-surfactant inclusion complexes, which results in a gradual decrease in the concentration of effectively available surfactant with increasing 2HPßCD concentration.

Preparation of calcium alginate nanoparticles using water-in-oil (W/O) nanoemulsions.

A procedure for the preparation of calcium alginate nanoparticles in the aqueous phase of water-in-oil (W/O) nanoemulsions was developed. The emulsions were produced from mixtures of the nonionic surfactant tetraethylene glycol monododecyl ether (C(12)E(4)), decane, and aqueous solutions of up to 2 wt % sodium alginate by means of the phase inversion temperature (PIT) emulsification method. This method allows the preparation of finely dispersed emulsions without a large input of mechanical energy. With alginate concentrations of 1-2 wt % in the aqueous phase, emulsions showed good stability against Ostwald ripening and narrow, monomodal distributions of droplets with radii <100 nm. Gelation of the alginate was induced by the addition of aqueous CaCl(2) to the emulsions under stirring, and particles formed were collected using a simple procedure based on extraction of the surfactant on addition of excess oil. The final particles were characterized using cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). They were found to be essentially spherical with a homogeneous interior, and their size was similar to that of the initial emulsion droplets. The herein presented "low-energy" method for preparation of biocompatible nanoparticles has the potential to be used in various applications, e.g., for the encapsulation of sensitive biomacromolecules.

Inclusion of a single-tail amino acid-based amphiphile in a lipoplex formulation: effects on transfection efficiency and physicochemical properties.

Effects of the addition of a cationic amino acid-based synthetic amphiphile, arginine N-lauroyl amide dihydrochloride (ALA), to a lipid-based transfection formulation have been investigated. It is shown that the inclusion of ALA results in a substantial enhancement of the transfection capability of lipoplexes prepared with liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine and cholesterol, which themselves mediate highly efficient transfection. A possible explanation for the increased biological activity is that ALA adsorbed to the surface of the DNA-lipid complexes is involved in triggering internalization. However, in order to identify possible additional factors underlying the enhanced transfection efficiency, the physical properties of formulations with and without ALA were characterized using cryo-transmission electron microscopy, dynamic light scattering, and an ethidium bromide intercalation assay. ALA seems to have limited influence on the initial internal structure of the complexes and the protection of DNA, but its presence is found to decrease the average effective size of the dispersed particles; this change in size may be important in improving the biological activity. Furthermore, ALA can act to influence the transfection efficiency of the formulation by promoting the release of DNA following internalization in the transfected cells.

Size and morphology of assemblies formed by DNA and lysozyme in dilute aqueous mixtures.

Assemblies formed by a well-defined quality of DNA (4331 bp T7 DNA) and the small net-cationic protein lysozyme in dilute aqueous solutions have been characterized using cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) as the main techniques. In a wide range of different DNA to lysozyme ratios in solutions of low ionic strength, dispersions of aggregates with the same general morphology and a practically constant hydrodynamic size are formed. The basic structure formed in the dispersions is that of rather flexible worm-like assemblies with a diameter of 10-20 nm, which are suggested to be made up by bundles of on the order of 10 DNA chains with an intervening matrix of lysozyme. With increased ionic strength, the worm-like appearance of the assemblies is lost and they adopt a less well-defined shape. The results suggest that the formation of the DNA-lysozyme aggregates is strongly influenced by cooperative assembly of the components and that, in addition to the electrostatic attraction between DNA and lysozyme, attractive interactions between the protein units are important in governing the behavior of the system.

Interactions between DNA and nonionic ethylene oxide surfactants are predominantly repulsive.

In the present work, the interactions between double-stranded (ds) or single-stranded (ss) DNA and nonionic ethylene oxide (EO) surfactants, with special attention to the possible contributions from hydrophobic interactions, have been investigated using a multitechnique approach. It was found that the presence of ss as well as dsDNA induces a slight decrease of the cloud point of pentaethylene glycol monododecyl ether (C(12)E(5)). Assessment of the partitioning of DNA between the surfactant-rich and surfactant-poor phases formed above the cloud point showed that the polymer was preferably located in the surfactant-poor phase. Surface tensiometry experiments revealed that neither of the DNA forms induced surfactant micellization. Finally, it was shown by DNA melting measurements that another EO surfactant (C(12)E(8)) did not affect the relative stabilities of ss and dsDNA. To summarize, all experiments suggest that the net interaction between DNA and nonionic surfactants of the EO type is weakly repulsive, which can be attributed mainly to steric effects. In general, the results were practically identical for the ds and ss forms of DNA, except those from the cloud point experiments, where the decrease of the cloud point was less pronounced with ssDNA. This finding indicates the presence of an attractive component in the interaction, which can reasonably be ascribed to hydrophobic effects.

Interactions between cationic lipid bilayers and model chromatin.

Complexes formed in mixtures of cationic liposomes of varying charge density and nucleosome core particles (NCPs) or nucleosome arrays have been characterized. Under most of the conditions studied, the lipids and NCPs or arrays formed lamellar structures similar to those obtained with the liposomes and pure DNA. Thus, the dissociation of DNA from the NCP or nucleosome array and the formation of a DNA-lipid complex is thermodynamically favored, which can likely be ascribed mainly to the gain in entropy on release of the small counterions. Only at very low liposome charge densities are there indications that the NCPs/arrays do not dissociate upon interaction with the lipid bilayers. The reported results can serve as a valuable reference point in investigations of biologically more relevant systems.

Structural evolution of oleyl betainate aggregates: in situ formation of small unilamellar vesicles.

Betaine esters prepared from long-chain alcohols are a class of hydrolyzable cationic surfactants that is interesting both because the compounds can be designed to give harmless products on degradation and that the hydrolysis products can induce potentially useful changes in the properties of systems where such surfactants are present. In this work, the evolution in structure of aggregates formed by oleyl betainate during hydrolysis of the compound has been investigated using (1)H NMR and cryo-transmission electron microscopy (cryo-TEM). With an increasing extent of hydrolysis, and thus an increasing fraction of oleyl alcohol in the aggregates, the aggregate structure changes in a sequence consistent with an increase in the average packing parameter of the surfactant-alcohol mixture, from spherical micelles, via wormlike micelles, to vesicles. An important result from this work is that it demonstrates a means of in situ production of small unilamellar vesicles with a rather narrow size distribution.

Phase behavior and coassembly of DNA and lysozyme in dilute aqueous mixtures: a model investigation of DNA-protein interactions.

Results from an experimental investigation of the phase behavior of an aqueous system of DNA from salmon testes and the protein lysozyme are presented. At very low concentrations of either or both of the macromolecular components, wormlike assemblies with a width of the order of 10 nm are formed. There are strong indications that direct interactions between the protein units are instrumental both in driving the phase separation and in controlling the morphology of the formed assemblies.

Dissolution and gelation of cellulose in TBAF/DMSO solutions: the roles of fluoride ions and water.

Solutions of cellulose in a mixture of tetrabutylammonium fluoride and dimethyl sulfoxide (TBAF/DMSO) containing small and varying amounts of water were studied by nuclear magnetic resonance (NMR). By measuring the composition dependences of (19)F NMR and (1)H NMR chemical shifts and line widths, details on the dissolution and gelation mechanisms for cellulose in TBAF/DMSO were elucidated. Our results suggest that the strongly electronegative fluoride ions act as hydrogen bond acceptors to cellulose hydroxyl groups, thus dissolving the polymer by breaking the cellulose-cellulose hydrogen bonds and by rendering the chains an effective negative charge. It was found that the fluoride ions also interact strongly with water. Small amounts of water remove the fluoride ions from the cellulose chains and allow reformation of the cellulose-cellulose hydrogen bonds, which leads to formation of highly viscous solutions or gels even at low cellulose concentrations.

Self-assembly of cationic surfactants that contain thioether groups in the hydrophobic tails.

Self-assembly in aqueous solutions of cationic surfactants that carry thioether groups in their hydrophobic tails has been investigated. Of particular interest was the identification of possible changes in the aggregate structure due to the presence of sulfur atoms. Solutions of four different compounds [CH(3)CH(2)S(CH(2))(10)N(CH(3))(3)(+)Br(-) (2-10), CH(3)(CH(2))(5)S(CH(2))(6)N(CH(3))(3)(+)Br(-) (6-6), CH(3)(CH(2))(7)S(CH(2))(6)N(CH(3))(3)(+)Br(-) (8-6), and CH(3)(CH(2))(7)S(CH(2))(8)N(CH(3))(3)(+)Br(-) (8-8)] were characterized by (1)H NMR, (13)C NMR, NMR diffusometry, and conductivity measurements. In addition to investigating aqueous solutions containing each of the thioethers present as the sole solute, mixtures of 2-10 or 6-6 with dodecyltrimethylammonium bromide (DTAB) were studied. The addition of a sulfide group to the hydrophobic tail causes an increase in the critical micelle concentration but has a limited effect on the aggregate structure. Micelles are formed at a well-defined concentration for all of the investigated surfactants and surfactant mixtures. However, a comparison of the behavior of concentrated solutions of 8-8 to that of solutions of hexadecyltrimethylammonium bromide (CTAB) of similar concentrations suggests that the presence of a sulfur atom decreases the tendency for micellar growth. This may be a consequence of a slightly higher preference for the micellar surface of a sulfur atom as compared to that of a methylene group in a similar position, an idea that is also supported by results for the surfactant mixtures.

Unusual aqueous-phase behavior of cationic amphiphiles with hydrogen-bonding headgroups.

Two cationic surfactants with hydroxyl and carbamate hydrogen-bonding sites at their headgroups were synthesized. Both surfactants are ionic liquids (one of them at room temperature). Samples are isotropic solutions over the entire 0-100% concentration range, which is highly unusual for ionic surfactants. Surface tension, NMR, and conductivity measurements indicate classical micelle formation in aqueous solutions with CMCs below 10 mM. Pulse-gradient spin-echo (PGSE) NMR confirms micelle formation and provides micellar hydrodynamic radii of about 3.8 nm. Because this value is larger than the length of the extended surfactant molecules, about 2.7 nm, it appears that hydrogen-bonded water of hydration contributes substantially to the effective micelle size. At higher concentrations (above 25 wt %), surfactant solutions become viscous, but line broadening in the NMR is small relative to that found with a conventional cationic surfactant (CTAB). Thus, long rod formation, the source of line broadening in the latter, is absent with the new surfactants. Finally, PGSE NMR data show a 5-fold decrease in the diffusion coefficient between 5 and 20 wt %, above which the diffusion coefficients remain constant. The results are best explained by micelle clustering that is likely aided by intermicellar hydrogen bonding. The possibility of an isotropic liquid crystal (LC) phase with cubic symmetry is discussed and dismissed, demonstrating that LC formation of ionic surfactants at high concentrations, the usual behavior in past work, need not occur. Nor is there a definite connection between ionic liquid behavior and isotropic morphology.

Studies on an ester-modified cationic amphiphile in aqueous systems: behavior of binary solutions and ternary mixtures with conventional surfactants.

The aqueous behavior of an ester-modified cationic amphiphile with the molecular structure CH3CH2O(C=O)(CH2)6(C=O)O(CH2)8N+(CH3)3Br-, in the following referred to as A, has been investigated. Systems with A as the only solute, as well as different aqueous mixtures with conventional cationic surfactants, primarily dodecyltrimethylammonium bromide (DTAB), were included in the study. Isotropic solution samples were characterized using 1H NMR, 13C NMR, NMR diffusometry, and conductivity measurements, whereas liquid crystalline samples were investigated by optical polarization microscopy and small-angle X-ray diffraction. The results are compared to the behavior of the binary system of DTAB and water. A does not exhibit a typical surfactant behavior. When it is present as the only solute in a binary aqueous system, it forms neither conventional micelles nor liquid crystalline phases. However, there is clear evidence that it assembles with lower cooperativity into loose clusters at concentrations above 25-30 mM. When A is mixed with DTAB in solution, the two amphiphiles form mixed assemblies, the structure of which varies with the total amphiphile concentration. In concentrated mixtures with alkyltrimethylammonium surfactants, A can participate in hexagonal liquid crystalline phases even when it constitutes a significant fraction of the total amphiphile content.