Micellar-like Structures Research Articles

An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

Synthesis, crystal structure and Hirshfeld surface analysis of 1-(12-bromo-dodec-yl)indoline-2,3-dione.

In the title compound, C20H28BrNO2, the indoline portion is almost planar and the 12-bromo-dodecyl chain adopts an all-trans conformation apart from the gauche terminal C-C-C-Br fragment. A micellar-like structure is generated in the crystal by C-H⋯O hydrogen bonds and π-stacking inter-actions between indolinedione head groups and inter-calation of the 12-bromo-dodecyl tails. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (58.9%), H⋯O/O⋯H (17.9%) and H⋯Br/Br⋯H (9.5%) contacts. A density functional theory (DFT) optimized structure at the B3LYP/ 6-311 G(d,p) level shows good agreement with the experimentally determined mol-ecular structure in the solid state.

Micro-characterization of modified microemulsions loaded with gossypol, pure and extracted from cottonseed

Microemulsions (MEs) have gained increasing interest as carriers of hydrophobic bioactives in the last decades. However, it is still difficult to control the uptake and the release of bioactives directly extracted from plants. In this study, modified ME nanodroplets (nano-sized self-assembled liquids, NSSLs) were employed as extraction medium of gossypol, a toxic component of cottonseed. Loading was performed using both pure gossypol, and gossypol obtained by extraction from cottonseed. We achieved two goals: i) remove gossypol from cottonseed to obtain cotton-oil free of gossypol; and ii) extract gossypol directly into a nano-delivery vehicle for biomedical purposes. Structural and dynamical information on the unloaded and gossypol-loaded NSSL systems were obtained by self-diffusion nuclear magnetic resonance, SD-NMR, and spin-probe electron paramagnetic resonance (EPR) studies. The results showed that NSSL formed fluid water-in-oil (W/O) nano domains at the lowest water contents; a more viscous bicontinuous structure at comparable oil and water contents, and, finally, oil-in-water (O/W, micellar-like) at the higher concentration of water. These micellar-like structures were more fluid at the external hydrated surface, as demonstrated by SD-NMR, while the lipidic region tested by EPR revealed an increasing packing. In all these structures, gossypol mainly localized in the lipophilic region close to the water interface. Overall, SD-NMR and EPR provided complementary information, helping to clarify the structural properties of NSSLs formed at different water contents and their ability to incorporate gossypol also directly from cottonseed-NSSL mixtures.

Soluble Polymer Complexes of Ceftriaxone and Cefotaxime with Cellulose Sulfate Acetate Salt Forms and Their Adsorption on Carbon Sorbents

Effect of the nature of a counterion in the salt forms of cellulose sulfate acetate on the composition, hydrodynamic characteristics, surface activity, and sizes of supramolecular structures of water-soluble complexes with ceftriaxone and cefotaxime is studied. The relationship between the position of counterion in the lyotropic series of alkali-metal cations and the properties of complexes is ascertained. It is shown that the amount of the bound antibiotic decreases in the sequence K+–Na+–Li+. The sizes of micellar-like supramolecular structures in solution and in the solid phase (films cast from solution) change in a similar manner. These structures can form because of hydrophobization of the macromolecular chain of polyelectrolyte. It is found that the adsorption of ceftriaxone and cefotaxime polymer complexes on various carbon sorbents depends on the porous structure of the activated carbon and makes it possible to provide the transfer of antibiotic to the small intestine in the unchanged form. This opens the possibility to create a new tablet dosage form of cephalosporin antibiotics while fully preserving their pharmacological activity.

Cyclodextrins: structure, physicochemical properties and pharmaceutical applications

Since their discovery over 100 years ago cyclodextrins (CDs) have been the subject of numerous scientific publications. In 2016 alone CDs were the subject of over 2200 research articles published in peer-reviewed journals and mentioned in over 2300 patents and patent applications, many of which were on pharmaceutical applications. Natural CDs and their derivatives are used as enabling pharmaceutical excipients that enhance aqueous solubility of poorly soluble drugs, increase drug permeability through biological membranes and improve drug bioavailability. Unlike conventional penetration enhancers, their hydrophilic structure and high molecular weight prevents them from penetrate into lipophilic membranes leaving biological membranes intact. The natural CDs and some of their derivatives have monographs in pharmacopeias and are also commonly used as food additives and in toiletry products. CDs form inclusion complexes with lipophilic moieties of hydrophobic drugs. Furthermore, CDs are able to form non-inclusion complexes and self-assembled aggregates; small and large complex aggregates with micellar-like structures that can enhance drug solubility. Excipients commonly used in pharmaceutical formulations may have additive or inhibiting effect on the CD solubilization. Here various methods used to investigate CD aggregate formation are reviewed as well as techniques that are used to increase the solubilizing effects of CDs; methods that enhance the apparent intrinsic solubility of drugs and/or the complexation efficacy and decrease the amount of CD needed to develop CD-containing pharmaceutical formulations. It will be explained how too much or too little CD can hamper drug bioavailability, and the role of CDs in solid dosage forms and parenteral formulations, and examples given on how CDs can enhance drug delivery after ocular, nasal and pulmonary administration.

Synthesis and Investigation of Double Stimuli-Responsive Behavior of N-Isopropylacrylamide and Maleic Acid Copolymer in Solutions

Poly(N-isopropylacrylamide-co-maleic acid) [poly(NIPAAm-co-MA)] linear copolymer with comonomer molar ratio NIPAAm:MA = 94:6 was synthesized by free-radical copolymerization. The molar mass, MsD = 28,000 Da, of poly(NIPAAm-co-MA) was determined using hydrodynamic methods. The self-assembly of poly(NIPAAm-co-MA) in aqueous solution at a concentration of 0.015 g cm−3 within the pH interval from 1.8 to 10.6 and the temperature interval from 22 to 60°C was investigated by static and dynamic light scattering. The copolymer showed a double temperature and pH responsiveness. Three types of particles, namely, macromolecules (or unimers), micellar-like structures, and loose aggregates existed in the poly(NIPAAm-co-MA) aqueous solutions. The fraction of dissolved entities and the hydrodynamic radii of the micellar-like structures and loose aggregates depended considerably on temperature and pH. The temperature of the phase separation and the width of the phase separation interval increased with pH. An influence of pH on kinetic processes in poly(NIPAAm-co-MA) solutions was observed.

Self-assembly of POSS-containing block copolymers: Fixing the hierarchical structure in networks

A series of P(MMA-co-GMA)-b-PMAPOSS block copolymers (BCPs) were synthesized by ATRP. The BCPs contain POSS (polyhedral oligomeric silsesquioxane) as a pendant unit on a particular polymer block chain. In selective solvents, the BCPs self-assemble to form ordered micellar-like structures. Spherical, cylindrical or vesicle-like morphologies were produced by tuning the BCP and mixed solvent compositions. Crosslinking of the BCP by the reaction of the glycidyl groups in the P(MMA-co-GMA) block of the micelle shell (GMA = glycidyl methacrylate) with a diamine results in a long-range structure ordering. The hexagonally packed cylindrical arrangement of the BCP networks was revealed by SAXS and TEM. The hierarchical, ordered structure of the POSS-containing hybrids was fixed by crosslinking. In addition, transient physical gels were prepared from triblock copolymers with outer MAPOSS blocks, and their rheological behavior was investigated.

Unravelling the ultrastructure of ascending colon fluids from patients with ulcerative colitis by cryogenic transmission electron microscopy

Studies on the characterization of ascending colonic fluids are scarce, limited to physicochemical characterization of their composition, and little is known for the morphology of the produced colloidal phases. In an attempt to gain insights for their structure at the ultrastructural level, samples from the lumen of ascending colon were collected from patients with ulcerative colitis in remission. After ultracentrifugation, the supernatants of two samples (one with high and one with low cholesterol level) were visualized by means of cryogenic transmission electron microscopy. In the supernatants with high cholesterol content, micellar-like structures, bilayer fragments, open vesicles, and uni-, bi- and trilamellar vesicles were abundant. In addition, crystals of cholesterol were frequently observed. In contrast, in the sample with low cholesterol content, oily solids, plates of cholesterol monohydrate and elongated structures were present. Few unilamellar vesicles were occasionally visualized. The current study gives direct evidence, for the first time, of the existence of 'remnants' of lipolytic products in the fasted ascending colon. The impact of these structures to colonic absorption of drugs is an open question.

Hierarchical Organization of Purely Peptidic Amphiphiles into Peptide Beads

A broad range of new properties is emerging from supramolecular aggregates as they pass beyond the limitations of simple molecules. Self-assembled structures of purely peptidic amphiphiles may exploit such properties to produce biocompatible, smart materials for drug administration. In aqueous media, the solid-phase derived amphiphilic undecapeptide described herein (Ac-X3-gT) forms self-assembled particles of spherical shape with diameters between 200 and 1500 nm, termed “peptide beads”. The beads result from hierarchical organization of micellar-like structures, a fact determined by a combination of investigations carried out by electron and atomic force microscopy (AFM), static and dynamic light scattering, and small-angle X-ray scattering. These highly ordered structures agree with the concept of multicompartmentization and represent the first example of supramicellar assemblies based purely on peptides. New structural insights, as presented here, allow a better understanding of the beads’ capacity to embed hydrophobic and hydrophilic payloads and therefore provide new perspectives for drug delivery applications that may result from this new class of material.

Self-assembled nanostructures in mixed anionic–neutral double hydrophilic block copolymer/cationic vesicle-forming surfactant solutions

The self-assembled nanostructures formed in mixed solutions of a double hydrophilic anionic–neutral block copolymer, poly[(2-sulfamate-3-carboxylate)isoprene-b-ethylene oxide] (SCIEO), and the vesicle-forming surfactant, didodecyldimethylammonium bromide (DDAB), are investigated. In these solutions electrostatic interactions exist between the anionic poly[(2-sulfamate-3-carboxylate)isoprene] block and the cationic surfactant. Combined static and dynamic light scattering measurements indicate that at low copolymer concentration vesicles of DDAB with adsorbed block copolymer chains are present in the solutions. As block copolymer concentration increases the structure of the nanoassemblies transforms to a core–shell, micellar-like structure, with a poly(ethylene oxide) corona and a core formed by the complex of poly[(2-sulfamate-3-carboxylate)isoprene] chains and DDAB molecules. This transformation of global aggregate structure should be attributed to a collapse of the DDAB vesicle bilayer as the number of complexed surfactant head groups to anionic polymeric segments increases. This happens in order to satisfy the conformational requirements of the complexing polymeric block and the steric requirements in the PEO corona, which apparently overcome the existing interactions between surfactant molecules in the ordered bilayer of initial DDAB vesicles. Block copolymer decorated vesicles show lower stability to an increase in solution temperature compared to micellar-like aggregates. Both nanostructures are found to be stable to changes in solution ionic strength, due to the combination of electrostatic and hydrophobic interactions acting within the mixed aggregates.

Photoresponsive polymersomes as smart, triggerable nanocarriers

The need for sophisticated systems to improve drug delivery to the body is growing, as is the complexity of therapeutic agents available to treat a variety of conditions. Among the requirements for intelligent drug delivery systems (DDS), responsiveness is highly desirable as a means to control pharmacokinetics and pharmacodynamics. Here, we study the potential of polymeric vesicles obtained from the self-assembly of a photocleavable amphiphilic block copolymer as a light-triggered DDS. The vesicles disintegrate upon UV irradiation, yielding small micellar-like structures, and simultaneously releasing their payload. The versatility of our system is tested both for low molecular weight molecules (fluorescein and ATTO655 dye), and for proteins (enhanced green fluorescent protein). By varying the UV intensity, the payload is released in a controlled manner, as established by fluorescence spectroscopy and fluorescence correlation spectroscopy. Therefore, these responsive polymer vesicles serve as smart, triggerable nanocarriers that can be applied to a variety of payloads, ranging from conventional drug molecules to proteins, enzymes, or DNA.

Impact of denaturation with urea on recombinant apolipoprotein A‐IMilano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I(Milano) (apo A-I(M)) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic alpha helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I(M) mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 x 10(-9) cm2/s in the absence of urea to 6.0 x 10(-8) cm2/s when apo A-I(M) is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.

Soluble complexes in aqueous mixtures of low charge density comb polyelectrolyte and oppositely charged surfactant probed by scattering and NMR

A low charge density polyelectrolyte with a high graft density of 45 units long poly(ethylene oxide) side-chains has been synthesized. In this comb polymer, denoted PEO 45MEMA:METAC-2, 2 mol% of the repeating methacrylate units in the polymer backbone carry a permanent positive charge and the remaining 98 mol% a 45 unit long PEO side-chain. Here we describe the solution conformation of this polymer and its association with an anionic surfactant, sodium dodecylsulfate, SDS. It will be shown that the polymer can be viewed as a stiff rod with a cross-section radius of gyration of 29 Å. The cross section of the rod contracts with increasing temperature due to decreased solvency of the PEO side-chains. The anionic surfactant associates to a significant degree with PEO 45MEMA:METAC-2 to form soluble complexes at all stoichiometries. A cooperative association is observed as the free SDS concentration approaches 7 mM. At saturation the number of SDS molecules associated with the polymer amounts to 10 for each PEO side-chain. Two distinct populations of associated surfactants are observed, one is suggested to be molecularly distributed over the comb polymer and the other constitutes small micellar-like structures at the periphery of the aggregate. These conclusions are reached based on results from small-angle neutron scattering, static light scattering, NMR, and surface tension measurements.

Manipulating the Solubility of Gold Nanoparticles Reversibly and Preparation of Water-Soluble Sphere Nanostructure through Micellar-like Solubilization

On the basis of micellar solubilization, a strategy tuning the solubility of gold nanoparticles reversibly was developed. Hydrophobic gold nanoparticles stabilized by octadecylamine (ODA-gold) solubilized in the micellar-like core of poly(vinylpyrrolidone) (PVP) in water to form gold nanoparticles with protective multilayer induced by reduction of interfacial energy. Interestingly, upon redispersing in chloroform, the PVP micellar-like structure can break down and the ODA-gold can be released again. By changing the ratio of PVP/ODA, size-controllable hydrophilic spherical assembly can be prepared. On the basis of the observation of transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR), a reasonable mechanism is interpreted thermodynamically.

Synthesis and Characterization of Multiblock Copolymers Based on Spider Dragline Silk Proteins

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.

Cationic Shape-Persistent Macrocycles: The Unexpected Formation of a Nano-Size Supramolecular Dimer

Shape-persistent macrocycles with a rigid pyridyl core and a flexible oligo-alkyl corona aggregate to some extent in nonpolar solvents to form large (tubular) aggregates. To increase the assembling tendency, the intraannular pyridyl groups of the macrocycles were alkylated. Unexpectedly, the quarternized macrocycles show no tendency at all to form tubular micellar-like structures but form well-defined dimers, as determined by X-ray and dynamic light scattering.

Phase and Gelation Behavior of 2‐Hydroxy‐3‐(N, N‐dimethyl‐N‐dodecylammonium)propyloxy Starches

AbstractSome starches containing quaternary dimethylalkylammonium groups exhibit an unique phase behavior. A solid phase or gel phase is formed upon cooling, i.e. they are temperature‐responsive polymers. The aim of this study was to investigate the phase and gelation behavior of hydrophobically modified quaternary ammonium starch ethers in aqueous solutions. The mechanisms behind the phase behavior and hydrophobic character were investigated by light scattering (turbidity) and rheological measurements. A relatively large increase in the complex viscosity at higher concentrations was observed when the solutions were cooled to room temperature. The phase angle decreased drastically at a certain critical temperature. The decrease in the phase angle depended on the concentration of starch in solution, higher concentrations showing the greatest decrease and lower concentrations showing no significant change. Turbidity measurements indicated that a solid‐like highly concentrated phase was precipitated. The starch with zero net charge showed a larger increase in turbidity than the starch with a positive net charge, which indicates that particular precipitation is favored by a zero net charge and that the formation of a gel network is favored by charged starch molecules.

Pharmacological effects of formulation vehicles : implications for cancer chemotherapy.

The non-ionic surfactants Cremophor EL (CrEL; polyoxyethyleneglycerol triricinoleate 35) and polysorbate 80 (Tween) 80; polyoxyethylene-sorbitan-20-monooleate) are widely used as drug formulation vehicles, including for the taxane anticancer agents paclitaxel and docetaxel. A wealth of recent experimental data has indicated that both solubilisers are biologically and pharmacologically active compounds, and their use as drug formulation vehicles has been implicated in clinically important adverse effects, including acute hypersensitivity reactions and peripheral neuropathy.CrEL and Tween 80 have also been demonstrated to influence the disposition of solubilised drugs that are administered intravenously. The overall resulting effect is a highly increased systemic drug exposure and a simultaneously decreased clearance, leading to alteration in the pharmacodynamic characteristics of the solubilised drug. Kinetic experiments revealed that this effect is primarily caused by reduced cellular uptake of the drug from large spherical micellar-like structures with a highly hydrophobic interior, which act as the principal carrier of circulating drug. Within the central blood compartment, this results in a profound alteration of drug accumulation in erythrocytes, thereby reducing the free drug fraction available for cellular partitioning and influencing drug distribution as well as elimination routes. The existence of CrEL and Tween 80 in blood as large polar micelles has also raised additional complexities in the case of combination chemotherapy regimens with taxanes, such that the disposition of several coadministered drugs, including anthracyclines and epipodophyllotoxins, is significantly altered. In contrast to the enhancing effects of Tween 80, addition of CrEL to the formulation of oral drug preparations seems to result in significantly diminished drug uptake and reduced circulating concentrations. The drawbacks presented by the presence of CrEL or Tween 80 in drug formulations have instigated extensive research to develop alternative delivery forms. Currently, several strategies are in progress to develop Tween 80- and CrEL-free formulations of docetaxel and paclitaxel, which are based on pharmaceutical (e.g. albumin nanoparticles, emulsions and liposomes), chemical (e.g. polyglutamates, analogues and prodrugs), or biological (e.g. oral drug administration) strategies. These continued investigations should eventually lead to more rational and selective chemotherapeutic treatment.

Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application

A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1–5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.

Interaction between a Novel Gemini Surfactant and Cyclodextrin: NMR and Surface Tension Studies

The interaction between cyclodextrins, hydroxypropyl-β-cyclodextrin (HPβCD), and hydroxypropyl-γ-cyclodextrin (HPγCD) and a novel type of nonionic surfactant synthesized from a fatty acid has been investigated. The so-called nonionic heterogemini surfactant (NIHG750) contains two hydrophobic groups and two hydrophilic groups, composed of one monomethyl ethylene glycol and one secondary OH group, CH3(CH2)7–CH[OH]–CH[O(CH2CH2O)16CH3]–(CH2)7CN. Surface tension studies indicate that micelles form in NIHG750 systems in both the presence and the absence of small quantities (molar ratio (HPβCD:NIHG750)≈2) of cyclodextrin (HPβCD or HPγCD). This gives NIHG surfactants an advantage compared to single-tailed nonionic surfactants, which generally lose their ability to micellize at much lower additions of cyclodextrins. However, the interaction between HPβCD and NIHG750 results in a disruption of the micellar aggregates at higher levels of cyclodextrin. In the dilute systems (CNIHG750<0.1% (w/w) aprox) prolate-shaped mixed aggregates (HPβCD and NIHG750) form, with a short and a long axis of the order of 8–9 and 17–20 Å, respectively. These gradually aggregate into micellar-like structures at higher concentrations. In the aqueous bulk phase HPβCD interacts mainly with the hydrophobic part of NIHG750, but both NMR and surface tension measurements indicate that an interaction with the hydrophilic part of NIHG750, as well, may exist. This interaction results in a better packing of NIHG750 at air-water interfaces. However, at elevated temperatures results from turbidity measurements indicate that NIHG750 and HPβCD interact mainly through the hydrophilic part of the surfactant; a decrease in the cloud point temperature is observed. The interaction of the larger cavity molecule, HPγCD, with NIHG750, on the other hand, seems to be relatively weak. The interaction, when present, most probably takes place through inclusion of the hydrophilic EO part of NIHG750. The results suggest that HPγCD in combination with NIHG750 is a better solubilizing system than with HPβCD.