Cryogenic Transmission Electron Microscopy Images Research Articles

Mechanism of crystalline self-assembly in aqueous medium: a combined cryo-TEM/kinetic study.

Understanding the crystallization of organic molecules is a long-standing challenge. Herein, a mechanistic study on the self-assembly of crystalline arrays in aqueous solution is presented. The crystalline arrays are assembled from perylene diimide (PDI) amphiphiles bearing a chiral N-acetyltyrosine side group connected to the PDI aromatic core. A kinetic study of the crystallization process was performed using circular dichroism spectroscopy combined with time-resolved cryogenic transmission electron microscopy (cryo-TEM) imaging of key points along the reaction coordinate, and molecular dynamics simulation of the initial stages of the assembly. The study reveals a complex self-assembly process starting from the formation of amorphous aggregates that are transformed into crystalline material through a nucleation-growth process. Activation parameters indicate the key role of desolvation along the assembly pathway. The insights from the kinetic study correlate well with the structural data from cryo-TEM imaging. Overall, the study reveals four stages of crystalline self-assembly: 1) collapse into amorphous aggregates; 2) nucleation as partial ordering; 3) crystal growth; and 4) fusion of smaller crystalline aggregates into large crystals. These studies indicate that the assembly process proceeds according to a two-step crystallization model, whereby initially formed amorphous material is reorganized into an ordered system. This process follows Ostwald's rule of stages, evolving through a series of intermediate phases prior to forming the final structure, thus providing an insight into the crystalline self-assembly process in aqueous medium.

Formation of worm-like micelles in mixed N-hexadecyl-N-methylpyrrolidinium bromide-based cationic surfactant and anionic surfactant systems.

Through the descriptive and rheological characterization of worm-like micelles formed by N-hexadecyl-N-methylpyrrolidinium bromide and sodium laurate, the formation and properties of the worm-like micelles were affected by the concentrations of sodium laurate and temperature. Additionally, cryogenic transmission electron microscopy images further validated the formation of worm-like micelles.

Effect of polyethylene glycol, alkyl, and oligonucleotide spacers on the binding, secondary structure, and self-assembly of fractalkine binding FKN-S2 aptamer-amphiphiles.

Previously we identified an aptamer, named FKN-S2, which binds the cell surface protein fractalkine with high affinity and specificity. In this paper a hydrophobic dialkyl C16 tail was added to the aptamer to create an aptamer-amphiphile. We investigated how the tail and a spacer molecule of varying length and hydrophobicity, inserted between the tail and the aptamer headgroup, affect the binding, structure, and self-assembly properties of the aptamer-amphiphile. We synthesized aptamer-amphiphiles with no spacer (NoSPR), polyethylene glycol (PEG4, PEG8, PEG24), alkyl (C12 and C24), or oligonucleotide (T10 and T5: 10 and 5 thymine, and A10: 10 adenine) spacers. The addition of the tail reduced the binding affinity of the aptamer-amphiphile over 7.5-fold compared to the free aptamer. The hydrophobic alkyl spacers resulted in the greatest loss of affinity, and the hydrophilic PEG spacers improved amphiphile affinity but did not restore it to that of the free aptamer. Interestingly, oligonucleotide spacers produced the highest affinity amphiphiles. Nucleotide composition did not affect affinity, however, as the T10 and A10 spacers had equal affinity. The oligonucleotide spacer amphiphiles had the highest affinity because the oligonucleotide spacer increased the affinity of free aptamer; the FKN-S2 aptamer plus the oligonucleotide spacer had a higher affinity than the free FKN-S2 aptamer. Circular dichroism (CD) spectroscopy and thermal melting studies indicated the aptamer forms a stem-loop and intramolecular G-quadruplex, and the tail strongly stabilized the formation of the G-quadruplex in a buffer. Cryogenic transmission electron microscopy (cryo-TEM) imaging showed the aptamer-amphiphiles, independent of the spacer used, self-assembled into micelles and nanotapes, flat bilayer structures that were often twisted. Finally, liposomes functionalized with the FKN-S2 amphiphile were incubated with fractalkine expressing cells, and the amount of binding was dependent on the concentration of the amphiphile on the liposome surface.

Aerosol Performance and Long-Term Stability of Surfactant-Associated Liposomal Ciprofloxacin Formulations with Modified Encapsulation and Release Properties

Previously, we showed that the encapsulation and release properties of a liposomal ciprofloxacin formulation could be modified post manufacture, by addition of surfactant in concert with osmotic swelling of the liposomes. This strategy may provide more flexibility and convenience than the alternative of manufacturing multiple batches of liposomes differing in composition to cover a wide range of release profiles. The goal of this study was to develop a surfactant-associated liposomal ciprofloxacin (CFI) formulation possessing good long-term stability which could be delivered as an inhaled aerosol. Preparations of 12.5 mg/ml CFI containing 0.4% polysorbate 20 were formulated between pH 4.7 and 5.5. These formulations, before and after mesh nebulization, and after refrigerated storage for up to 2 years, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. Within the narrower pH range of 4.9 to 5.2, these formulations retained their physicochemical stability after 2-year refrigerated storage, were robust to mesh nebulization, and formed respirable aerosols with a volume mean diameter (VMD) of 3.7 μm and a geometric standard deviation (GSD) of 1.7. This study demonstrates that it may be possible to provide a range of release profiles by simple addition of surfactant to a liposomal formulation post manufacture, and that these formulations may retain their physicochemical properties after long-term refrigerated storage and following aerosolization by mesh nebulizer.

Sugar-bile acid-based bolaamphiphiles: from scrolls to monodisperse single-walled tubules.

The introduction of a mannose residue on carbon 3 of lithocholic acid gives rise to an asymmetric and rigid bolaamphiphilic molecule, which self-assembles in water to form elongated tubular aggregates with an outer diameter of about 20 nm. These tubular structures display a temporal evolution, where the average tube diameter decreases with time, which can be followed by time-resolved small-angle X-ray scattering experiments. Cryogenic transmission electron microscopy images collected as a function of time show that at short times after preparation tubular scrolls are formed via the rolling of layers, after which a complex transformation of the scrolls into single-walled tubules takes place. At long time scales, a further evolution occurs where the tubules both elongate and become narrower. The observed self-assembly confirms the tendency of bile acids and their derivatives to form supramolecular aggregates with an ordered packing of the constituent molecules. It also demonstrates that scrolls can be formed as intermediate structures in the self-assembly process of monodisperse single-walled tubules.

Origins of Microstructural Transformations in Charged Vesicle Suspensions: The Crowding Hypothesis

It is observed that charged unilamellar vesicles in a suspension can spontaneously deflate and subsequently transition to form bilamellar vesicles, even in the absence of externally applied triggers such as salt or temperature gradients. We provide strong evidence that the driving force for this deflation-induced transition is the repulsive electrostatic pressure between charged vesicles in concentrated suspensions, above a critical effective volume fraction. We use volume fraction measurements and cryogenic transmission electron microscopy imaging to quantitatively follow both the macroscopic and microstructural time-evolution of cationic diC18:1 DEEDMAC vesicle suspensions at different surfactant and salt concentrations. A simple model is developed to estimate the extent of deflation of unilamellar vesicles caused by electrostatic interactions with neighboring vesicles. It is determined that when the effective volume fraction of the suspension exceeds a critical value, charged vesicles in a suspension can experience "crowding" due to overlap of their electrical double layers, which can result in deflation and subsequent microstructural transformations to reduce the effective volume fraction of the suspension. Ordinarily in polydisperse colloidal suspensions, particles interacting via a repulsive potential transform into a glassy state above a critical volume fraction. The behavior of charged vesicle suspensions reported in this paper thus represents a new mechanism for the relaxation of repulsive interactions in crowded situations.

A drag reducing surfactant threadlike micelle system with unusual rheological responses to pH

A pH-responsive threadlike micellar system was developed by mixing alkyl bis(2-hydroxyethyl)methylammonium chloride (EO12) and trans-o-coumaric acid (tOCA). The rheological response of this system to pH is unusual in that it has viscoelasticity at both high and low pH levels, while it shows water-like behaviors at medium pH. Cryogenic transmission electron microscopy (Cryo-TEM) images confirmed the presence of TLMs at pH 3.5 and pH 9.8. This system also had DR (drag reduction) capability at low and high pH. The unusual rheological and micellar responses of this system to pH are caused by the dual pKa’s of tOCA. 1H NMR and zeta potential results support this hypothesis.

Vitamin C-driven epirubicin loading into liposomes

The encapsulation of anticancer drugs in a liposome structure protects the drug during circulation and increases drug accumulation in the cancer tissue and antitumor activity while decreasing drug toxicity. This paper presents a new method of active drug loading based on a vitamin C pH/ion gradient. Formulations were characterized in terms of the following parameters: optimal external pH, time and drug-to-lipid ratio for the purpose of remote loading, and in vitro stability. In the case of the selected drug, epirubicin (EPI), its coencapsulation increases its anticancer activity through a possibly synergistic effect previously reported by other groups for a free nonencapsulated drug/vitamin C cocktail. The method also has another advantage over other remote-loading methods: it allows faster drug release through liposome destabilization at the tumor site, thanks to the very good solubility of the EPI vitamin C salt, as seen on cryogenic transmission electron microscopy images. This influences the drug-release process and increases the anticancer activity of the liposome formulation. The liposomes are characterized as stable, with very good pharmacokinetics (half-life 18.6 hours). The antitumor activity toward MCF-7 and 4T-1 breast cancer cells was higher in the case of EPI loaded via our gradient than via an ammonium sulfate gradient. Finally, the EPI liposomal formulation and the free drug were tested using the murine 4T-1 breast cancer model. The antitumor activity of the encapsulated drug was confirmed (tumor-growth inhibition over 40% from day 16 until the end of the experiment), and the free drug was shown to have no anticancer activity at the tested dose.

Submicron hybrid vesicles consisting of polymer–lipid and polymer–cholesterol blends

Hybrid polymer–lipid and polymer–cholesterol submicron unilamellar vesicles with a diameter of ∼200 nm based on poly(dimethylsiloxane)-block-poly(2-methyl-oxazoline) (PDMS-b-PMOXA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were prepared by using the film-hydration and extrusion method. The vesicles were extensively characterized via dynamic and static light scattering (DLS and SLS), dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy imaging and force spectroscopy (AFM). The combination of these techniques provides information about the vesicle size, shape and long-term stability as well as the mechanical properties. DC-FCCS proved the formation of hybrid vesicles containing both components. Cryo-TEM and AFM studies revealed that the presence of cholesterol in vesicle membranes leads to a higher packing density resulting in a reduced membrane thickness and a significantly increased bending stiffness compared to pure polymersomes and polymer–lipid vesicles.

Influence of Counterions on Lauric Acid Vesicles and Theoretical Consideration of Vesicle Stability

The counterions, including inorganic cations, Na(+) and Cs(+), and organic cation, (C(2)H(5))(4)N(+), influence the phase behavior and self-assembled structures of the lauric acid (LA) in water. Dissolving LA in NaOH, CsOH, and (C(2)H(5))(4)NOH (tetraethylammonium hydroxide, TeAOH) solutions, respectively, we observed that the three systems totally exhibited the same phase behavior, from birefringent L(α) phase/precipitates (P) → L(α) phase → L(α) phase/L(1) (micelles) → L(1). The temperature influence on phase behavior was investigated, and with an increase of temperature, we observed that less phase behavior change occurred in the systems of LA/CsOH/H(2)O and LA/TeAOH/H(2)O, while the phase behavior of the LA/NaOH/H(2)O system exhibited an obvious change. Cryogenic transmission electron microscopy (cryo-TEM) images demonstrated that the different microstructures of L(α) phase samples in the three systems existed. For the systems of LA/NaOH/H(2)O and LA/TeAOH/H(2)O, uni- and multilamellar vesicles coexist for L(α) phase samples, as both the morphology and size of these vesicles are polydisperse. The curvatures of the bilayer membranes of the two systems are considered to vary from positive, zero, and even negative. However, only spherically unilamellar vesicles exist in the system of LA/CsOH/H(2)O, indicating that the bilayers are more rigid than those in the LA/NaOH/H(2)O and LA/TeAOH/H(2)O systems. Through the combination of the Helfrich curvature energy theory and the mass-action model, the effective bending constant K = 0.5 k(B)T in the LA/CsOH/H(2)O system was obtained, demonstrating that the unilamellar vesicles are stabilized by thermal fluctuations. A primary discussion for the effect of the nature of counterions on the stability and deformation of the vesicles is presented.

SPANosomes as Delivery Vehicles for Small Interfering RNA (siRNA)

Nonionic surfactant vesicles, or SPANosomes (SPs), comprised of cationic lipid and sorbitan monooleate (Span 80) were synthesized and evaluated as small interfering RNA (siRNA) vectors. The SPs had a mean diameter of less than 100 nm and exhibited excellent colloidal stability. The SP/siRNA complexes possessed a slightly positive zeta potential of 12 mV and demonstrated a high siRNA incorporation efficiency of greater than 80%. Cryogenic transmission electron microscopy (cryo-TEM) imaging of the SP/siRNA indicated a predominantly core-shell structure. The SP/siRNA complexes were shown to efficiently and specifically silence expression of both green fluorescent protein (GFP) (66% knockdown) and aromatase (77% knockdown) genes in breast cancer cell lines. In addition, the cellular trafficking pathway of the SP/siRNA was investigated by confocal microscopy using molecular beacons as probes for cytosolic delivery. The results showed efficient endosomal escape and cytosolic delivery of the siRNA cargo following internalization of the SP/siRNA complexes. In conclusion, Span 80 is a potent helper lipid, and the SPs are promising vehicles for siRNA delivery.

Reversible binding of an inorganic cluster-anion to the surface of a gold nanoparticle

Reversible dissociation of POM ligands from electrostatically stabilized monolayer assemblies on metal(0)-nanoparticle surfaces would provide substrates with direct access to reactive metal(0) lattices. As a result, POM-ligand lability could play an important role in catalysis by POM-monolayer protected metal(0) nanoparticles. POM-ligand lability was addressed by first obtained cryogenic transmission electron microscopy (cryo-TEM) images of monolayers of α - AlW 11 O 39 9 - on a 5.4 nm diameter gold-core nanoparticle. These are the smallest nanoparticles on which POM monolayers have been imaged by cryo-TEM. The binding affinities of α - K 9 AlW 11 O 39 (K 9 1) and α - Na 5 AlW 12 O 40 (Na 5 2) were obtained from Langmuir isotherms, and the relative labilities of the two ligands were assessed by UV–Vis analysis of changes in surface plasmon resonance (SPR) upon incremental additions of citrate. Thermodynamic constants, K, for monolayer formation by 1 and 2 are 20,000 and 150 M −1, respectively. In timed reactions with citrate, 1 exhibits a substantial kinetic barrier to dissociation, while 2 is rapidly exchanged by the organic protecting ligand.

Irreversible nanogel formation in surfactant solutions by microporous flow

Self-assembly of surfactant molecules into micelles of various shapes and forms has been extensively used to synthesize soft nanomaterials. Translucent solutions containing rod-like surfactant micelles can self-organize under flow to form viscoelastic gels. This flow-induced structure (FIS) formation has excited much fundamental research and pragmatic interest as a cost-effective manufacturing route for active nanomaterials. However, its practical impact has been very limited because all reported FIS transitions are reversible because the gel disintegrates soon after flow stoppage. We present a new microfluidics-assisted robust laminar-flow process, which allows for the generation of extension rates many orders of magnitude greater than is realizable in conventional devices, to produce purely flow-induced permanent nanogels. Cryogenic transmission electron microscopy imaging of the gel reveals a partially aligned micelle network. The critical flow rate for gel formation is consistent with the Turner-Cates fusion mechanism, proposed originally to explain reversible FIS formation in rod-like micelle solutions.

Hydrogen-Bond-Induced Microstructural Transition of Ionic Micelles in the Presence of Neutral Naphthols: pH Dependent Morphology and Location of Surface Activity

The effect of naphthols and methoxynaphthalenes on the microstructure transition of cetyltrimethylammonium bromide (CTAB) micelles is studied. Although the surface activities of these two types of organic dopants are strong, methoxynaphthalenes failed to promote spherical to worm-like micellar transition and to impart viscoelasticity to the aqueous CTAB solution, presumably due to their inability to form unique H-bonds with interfacial water. The micropolarity of OH sites of micelle-embedded naphthols is measured by observing the pK(a) shift at the micellar surface relative to bulk water. On the basis of spectroscopic and other data, the microstructures formed by both classes of dopants at the micellar surface are predicted. On the basis of hydroxyaromatic dopants, a simple and effective route to design pH-responsive viscoelastic worm-like micelles and the vesicles of single tail cationic surfactant (CTAB) is reported. Results are confirmed by observing cryogenic transmission electron microscopy (cryo-TEM) images.

Properties of microemulsions based on mixed nonionic surfactants and mixed oils

The systems studied were water/sucrose laurate/ethoxylated mono-di-glyceride/isopropylmyristate/peppermint oil. The solubilization capacity of water in the oils is dependent on the surfactants and oils mixing ratios (w/w). The transport properties (electrical conductivity and dynamic viscosity) were studied as function of water volume fraction. Electric percolation phenomenon was observed in these systems and the water volume fraction percolation thresholds were determined. The diffusion properties investigated by nuclear magnetic resonance confirm a progressive transformation of the water-in-oil to bicontinuous and inversion to oil-in-water microemulsions that occur upon dilution with water. The diffusion coefficients of surfactants increase with the increase in the water volume fraction. The structural parameters studied by small angle X-ray scattering that include the periodicity and correlation length were estimated. The periodicity increases linearly with the increase in the water volume fraction whereas the correlation length increases with the increase in the water volume fraction to a certain value then decreases. Cryogenic transmission electron microscopy images for diluted microemulsions revealed the presence of spheroidal droplets of up to 10 nm diameter.

Self-Assembly of Janus Cylinders into Hierarchical Superstructures

We present in-depth studies of the size tunability and the self-assembly behavior of Janus cylinders possessing a phase segregation into two hemicylinders. The cylinders are prepared by cross-linking the lamella-cylinder morphology of a polystyrene-block-polybutadiene-block-poly(methyl methacrylate) block terpolymer. The length of the Janus cylinders can be adjusted by both the amplitude and the duration of a sonication treatment from the micro- to the nanometer length. The corona segregation into a biphasic particle is evidenced by selective staining of the PS domains with RuO(4) and subsequent imaging. The self-assembly behavior of these facial amphiphiles on different length scales is investigated combining dynamic light scattering (DLS), small-angle neutron scattering (SANS), and imaging procedures. Cryogenic transmission electron microscopy images of the Janus cylinders in THF, which is a good solvent for both blocks, exhibit unimolecularly dissolved Janus cylinders with a core-corona structure. These results are corroborated by SANS measurements. Supramolecular aggregation takes place in acetone, which is a nonsolvent for polystyrene, leading to the observation of fiber-like aggregates. The length of these fibers depends on the concentration of the solution. A critical aggregation concentration is found, under which unimolecularly dissolved Janus cylinders exist. The fibers are composed of 2-4 Janus cylinders, shielding the inner insoluble polystyrene hemicylinder against the solvent. Herein, the SANS data reveal a core-shell structure of the aggregates. Upon deposition of the Janus cylinders from more concentrated solution, a second type of superstructure is formed on a significantly larger length scale. The Janus cylinders form fibrillar networks, in which the pore size depends on the concentration and deposition time of the sample.

Structural and Relaxometric Characterization of Peptide Aggregates Containing Gadolinium Complexes as Potential Selective Contrast Agents in MRI

The structural and relaxometric characterization of a novel class of supramolecular aggregates, as potential tumor-specific contrast agents in magnetic resonance imaging (MRI), is reported. The aggregates are based on a new monomer with an upsilon shape (MonY) that contains, in the same molecule, all three fundamental tasks that are required: 1) a hydrophobic moiety that allows the formation of supramolecular aggregates; 2) the bioactive CCK8 peptide for target recognition; and 3) a chelating agent able to give stable gadolinium complexes. As indicated by dynamic light scattering and small-angle neutron scattering (SANS) measurements, MonY and its gadolinium complex MonY(Gd) aggregate in aqueous solution to give ellipsoidal micelles with a ratio between the micellar axes of approximately 1.7 and an aggregation number N(agg) of approximately 30. There are no differences in the aggregation behavior of MonY and MonY(Gd), which indicates that the presence of metal ions, and therefore the reduction of the net charge, does not influence the aggregation behavior. When MonY or MonY(Gd) are blended with dioleoyl phosphatidylcholine (DOPC), the aggregation behavior is dictated by the tendency of DOPC to give liposomes. Only when the amount of MonY or MonY(Gd) is higher than 20 % is the coexistence of liposomes and micelles observed. The thickness d of the bilayer is estimated by SANS to be approximately 35-40 A, whereas cryogenic transmission electron microscopy images show that the diameter of the liposomes ranges from approximately 50 to 150 nm. Self-assembling micelles of MonY(Gd) present high relaxivity values (r(1p)=15.03 mM(-1) s(-1)) for each gadolinium complex in the aggregate. Liposomes containing MonY(Gd) inserted in the DOPC bilayer at a molar ratio of 20:80 present slightly lower relaxivity values (r(1p)=12.7 mM(-1) s(-1)), independently of their internal or external position in the liposome.

Differences in Cross-Link Chemistry between Rigid and Flexible Dithiol Molecules Revealed by Optical Studies of CdTe Quantum Dots

The cross-link chemistry of CdTe quantum dots (QDs) in solution is studied for different types of aliphatic (flexible) and aromatic (rigid) dithiol linker molecules. A remarkable difference in the cross-linking efficiency is observed: the rigid dithiols are shown to form aggregates at much lower concentrations. Qualitative and quantitative information on the formation of aggregates is obtained from cryogenic transmission electron microscopy (cryo-TEM) images and photoluminescence decay measurements. The luminescence decay curves are analyzed with a model for energy transfer to neighboring QDs in aggregates. The analysis shows that the cross-linking efficiency is 4 times higher for the rigid dithiols than for the flexible dithiols. The difference is attributed to the formation of loops for the flexible dithiols by attaching with both thiol groups to the same nanocrystal surface (preventing cross-linking), whereas the rigid aromatic dithiols cannot form loops and the second thiol group is oriented away from the surface (enabling cross-linking). The difference in conformation between flexible and rigid dithiols is confirmed by studies on the red-shift in the optical absorption spectra due to capping exchange of amines by monothiols or dithiols and by molecular simulations.

Computer‐vision determination of 3‐D geometric parameters of LDL particles via cryogenic transmission electron microscopy

Abstract— Previous research has shown that the size of the LDL macromolecules can have an effect on cardiovascular health and that LDL macromolecules may be non‐spherical in shape. Some of these studies, however, used methods that are not conducive to automatic determination of the 3‐D parameters of the particles. In particular, the prior methods used for determination of geometric‐parameter determination were either centrifugal separations or manual determination of parameters from cryogenic transmission electron micrographs. An application of computer‐vision techniques to automatically determine the 3‐D parameters from cryogenic transmission electron microscopy (CTEM) images will be described. Correlation of computer‐generated geometric models to the orthonormal projection CTEM imagery were investigated to determine the applicability of finding the pertinent geometric parameters of the expected discoid shape of the LDL particles. The processing showed that the discoid shape can be verified using small‐angle rotations that are more amenable to the limitations of CTEM imaging.

Photo-induced reduction of Noble metal ions to metal nanoparticles on tubular J-aggregates

Palladium and silver nanoparticles are formed on the surface of tubular J-aggregates of an amphiphilic tetrachlorobenzimidacarbocyanine dye by reduction of the respective metal cations in aqueous solution. Upon addition of the palladium complex Na2PdCl4to the aggregate solution, the absorption spectrum shows significant changes which is explained by partial destruction of the aggregates. Cryogenic transmission electron microscopy (cryo-TEM) images show that the tubular J-aggregates are randomly covered by well-separated Pd nanoparticles of approximately 1–3 nm size. Larger particles and higher particle density along the aggregates are obtained when an auxiliary reducing agent is added to the solution. The presence of the metallic particles leads to efficient fluorescence quenching giving clear evidence for super quenching. In similar experiments using AgNO3, silver nanoparticles are grown which are larger in size but less dense distributed along the aggregates. At least in the case of the silver particles, the spontaneous formation of metal nanoparticles is assumed to be initiated by a photo-induced electron transfer process (PET).