Liquid-liquid phase separation (LLPS) in DNA and chromatin systems from the perspective of colloid physical chemistry.

DNA is a highly charged polyelectrolyte and is prone to associative phase separation driven by the presence of multivalent cations, charged surfactants, proteins, polymers and colloids. The process of DNA phase separation induced by positively charged species is often called DNA condensation. Generally, it refers to either intramolecular DNA compaction (coil-globule transition) or intermolecular DNA aggregation with macroscopic phase separation, but the formation of a DNA liquid crystalline system is also displayed. This has traditionally been described by polyelectrolyte theory and qualitative (Flory-Huggins-based) polymer theory approaches. DNA in the cell nucleus is packed into chromatin wound around the histone octamer (a protein complex comprising two copies each of the four histone proteins H2A, H2B, H3 and H4) to form nucleosomes separated by linker DNA. During the last decade, the phenomenon of the formation of biomolecular condensates (dynamic droplets) by liquid-liquid phase separation (LLPS) has emerged as a generally important mechanism for the formation of membraneless organelles from proteins, nucleic acids and their complexes. DNA and chromatin droplet formation through LLPS has recently received much attention by in vitro as well as in vivo studies that established the importance of this for compartmentalisation in the cell nucleus. Here, we review DNA and chromatin LLPS from a general colloid physical chemistry perspective. We start with a general discussion of colloidal phase separation in aqueous solutions and review the original (pre-LLPS era) work on DNA (macroscopic) phase separation for simpler systems with DNA in the presence of multivalent cations and well-defined surfactants and colloids. Following that, we discuss and illustrate the similarities of such macroscopic phase separation with the general behaviour of LLPS droplet formation by associative phase separation for DNA-protein systems, including chromatin; we also note cases of segregative association. The review ends with a discussion of chromatin LLPS in vivo and its physiological significance.

Effect of polymer addition on the phase behavior of oil-water-surfactant systems of Winsor III type.

Ternary oil-water-surfactant systems can give rise to an O/W microemulsion in equilibrium with excess oil, a W/O microemulsion in equilibrium with excess water, or a bicontinuous microemulsion in equilibrium with excess oil and water. This type of phase behavior has been known for a long time and the three systems are often referred to as Winsor I, Winsor II and Winsor III, respectively after the British scientist P. A. Winsor who pioneered the area. The Winsor systems are technically important and well understood today. It was later found that addition of a polymer to the oil-water-surfactant system can influence the phase behavior considerably. While a hydrophilic polymer will be incorporated in the water phase and a hydrophobic polymer in the oil phase, an amphiphilic polymer with the right hydrophilic-lipophilic balance may expand the middle phase microemulsion in a Winsor III system. Expansion of the middle phase of such a system will lead to a reduction of the oil/microemulsion and the microemulsion/water interfacial tensions. This can be practically important, and the effect is currently of considerable interest for so-called surfactant flooding for enhanced oil recovery (EOR). Boosting the middle phase of the Winsor III system by addition of a polymer to the surfactant system is still not an established procedure and not so well understood from a scientific point of view. In this review we summarize the work done in the field and we demonstrate that the role of the polymer is intimately linked to its interactions with the three other components in the system: the oil, the water, and the surfactant(s).

On the formation and stability of cellulose-based emulsions in alkaline systems: Effect of the solvent quality.

With amphiphilic properties, cellulose molecules are expected to adsorb at the O/W interface and be capable of stabilizing emulsions. The effect of solvent quality on the formation and stability of cellulose-based O/W emulsions was evaluated in different alkaline systems: NaOH, NaOH-urea and tetrabutylammonium hydroxide (TBAH). The optimal solvency conditions for cellulose adsorption at the O/W interface were found for the alkaline solvent with an intermediate polarity (NaOH-urea), which is in line with the favorable conditions for adsorption of an amphiphilic polymer. A very good solvency (in TBAH) and the interfacial activity of the cation lead to lack of stability because of low cellulose adsorption. However, to achieve long-term stability and prevent oil separation in NaOH-urea systems, further reduction in cellulose's solvency was needed, which was achieved by a change in the pH of the emulsions, inducing the regeneration of cellulose at the surface of the oil droplets (in-situ regeneration).

Lignin enhances cellulose dissolution in cold alkali.

Aqueous sodium hydroxide solutions are extensively used as solvents for lignin in kraft pulping. These are also appealing systems for cellulose dissolution due to their inexpensiveness, ease to recycle and low toxicity. Cellulose dissolution occurs in a narrow concentration region and at low temperatures. Dissolution is often incomplete but additives, such as zinc oxide or urea, have been found to significantly improve cellulose dissolution. In this work, lignin was explored as a possible beneficial additive for cellulose dissolution. Lignin was found to improve cellulose dissolution in cold alkali, extending the NaOH concentration range to lower values. The regenerated cellulose material from the NaOH-lignin solvents was found to have a lower crystallinity and crystallite size than the samples prepared in the neat NaOH and NaOH-urea solvents. Beneficial lignin-cellulose interactions in solution state appear to be preserved under coagulation and regeneration, reducing the tendency of crystallization of cellulose.

Double-Chain Cationic Surfactants: Swelling, Structure, Phase Transitions and Additive Effects.

Double-chain amphiphilic compounds, including surfactants and lipids, have broad significance in applications like personal care and biology. A study on the phase structures and their transitions focusing on dioctadecyldimethylammonium chloride (DODAC), used inter alia in hair conditioners, is presented. The phase behaviour is dominated by two bilayer lamellar phases, Lß and Lα, with "solid" and "melted" alkyl chains, respectively. In particular, the study is focused on the effect of additives of different polarity on the phase transitions and structures. The main techniques used for investigation were differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS and WAXS). From the WAXS reflections, the distance between the alkyl chains in the bilayers was obtained, and from SAXS, the thicknesses of the surfactant and water layers. The Lα phase was found to have a bilayer structure, generally found for most surfactants; a Lß phase made up of bilayers with considerable chain tilting and interdigitation was also identified. Depending mainly on the polarity of the additives, their effects on the phase stabilities and structure vary. Compounds like urea have no significant effect, while fatty acids and fatty alcohols have significant effects, but which are quite different depending on the nonpolar part. In most cases, Lß and Lα phases exist over wide composition ranges; certain additives induce transitions to other phases, which include cubic, reversed hexagonal liquid crystals and bicontinuous liquid phases. For a system containing additives, which induce a significant lowering of the Lß-Lα transition, we identified the possibility of a triggered phase transition via dilution with water.

On the Development of All-Cellulose Capsules by Vesicle-Templated Layer-by-Layer Assembly.

Polymeric multilayer capsules formed by the Layer-by-Layer (LbL) technique are interesting candidates for the purposes of storage, encapsulation, and release of drugs and biomolecules for pharmaceutical and biomedical applications. In the current study, cellulose-based core-shell particles were developed via the LbL technique alternating two cellulose derivatives, anionic carboxymethylcellulose (CMC), and cationic quaternized hydroxyethylcellulose ethoxylate (QHECE), onto a cationic vesicular template made of didodecyldimethylammonium bromide (DDAB). The obtained capsules were characterized by dynamic light scattering (DLS), ζ potential measurements, and high-resolution scanning electron microscopy (HR-SEM). DLS measurements reveal that the size of the particles can be tuned from a hundred nanometers with a low polydispersity index (deposition of 2 layers) up to micrometer scale (deposition of 6 layers). Upon the deposition of each cellulose derivative, the particle charge is reversed, and pH is observed to considerably affect the process thus demonstrating the electrostatic driving force for LbL deposition. The HR-SEM characterization suggests that the shape of the core-shell particles formed is reminiscent of the spherical vesicle template. The development of biobased nano- and micro-containers by the alternating deposition of oppositely charged cellulose derivatives onto a vesicle template offers several advantages, such as simplicity, reproducibility, biocompatibility, low-cost, mild reaction conditions, and high controllability over particle size and composition of the shell.

Oral delivery of all-trans retinoic acid mediated by liposome carriers.

All-trans retinoic acid (ATRA) is a molecule that finds wide applications in medicine. Connection between cancer cell proliferation and ATRA is a well-established item. Driven by the potential applications of liposomes in stabilizing and protecting therapeutic compounds thus enabling effective delivery of encapsulated compounds, recent research efforts have been directed to understanding mechanisms of oral delivery through the gastrointestinal tract. The surface charge of the liposome bilayers can modify the interactions between the aggregates and the gastrointestinal fluids. Here, we investigated the ability of cationic and anionic liposomes to encapsulate, protect and deliver ATRA in an in-vitro digestion process as a different oral administration route. Stability and encapsulation efficiency of ATRA in negatively and positively charged liposomes enriched with α-tocopherol were investigated by means of UV-vis spectroscopy, dynamic light scattering and ζ-potential. The applicability of the carriers was tested by means of an in-vitro digestion procedure allowing for the measurement of the bioavailability of ATRA. From this study evidence was provided that the water insoluble molecules, ATRA and α-tocopherol are intercalated in liposome membranes regardless of the surface charge of the vesicle bilayers. Comparisons between cationic and anionic liposomes incorporating retinoic acid show differences in bioavailability. The cationic vesicles are preferable for a larger amount of ATRA bioavailability, which can be understood from electrostatic interactions. Thus ATRA is ionized in a wide range of pHs but protonated in anionic vesicles.

Hydrophobic interactions control the self-assembly of DNA and cellulose.

Desoxyribosenucleic acid, DNA, and cellulose molecules self-assemble in aqueous systems. This aggregation is the basis of the important functions of these biological macromolecules. Both DNA and cellulose have significant polar and nonpolar parts and there is a delicate balance between hydrophilic and hydrophobic interactions. The hydrophilic interactions related to net charges have been thoroughly studied and are well understood. On the other hand, the detailed roles of hydrogen bonding and hydrophobic interactions have remained controversial. It is found that the contributions of hydrophobic interactions in driving important processes, like the double-helix formation of DNA and the aqueous dissolution of cellulose, are dominating whereas the net contribution from hydrogen bonding is small. In reviewing the roles of different interactions for DNA and cellulose it is useful to compare with the self-assembly features of surfactants, the simplest case of amphiphilic molecules. Pertinent information on the amphiphilic character of cellulose and DNA can be obtained from the association with surfactants, as well as on modifying the hydrophobic interactions by additives.

Revisiting the dissolution of cellulose in H3PO4(aq) through cryo-TEM, PTssNMR and DWS.

Cellulose can be dissolved in concentrated acidic aqueous solvents forming extremely viscous solutions, and, in some cases, liquid crystalline phases. In this work, the concentrated phosphoric acid aqueous solvent is revisited implementing a set of advanced techniques, such as cryo-transmission electronic microscopy (cryo-TEM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR), and diffusing wave spectroscopy (DWS). Cryo-TEM images confirm that this solvent system is capable to efficiently dissolve cellulose. No cellulose particles, fibrils, or aggregates are visible. Conversely, PTssNMR revealed a dominant CP signal at 25 °C, characteristic of C-H bond reorientation with correlation time longer than 100 ns and/or order parameter above 0.5, which was ascribed to a transient gel-like network or an anisotropic liquid crystalline phase. Increasing the temperature leads to a gradual transition from CP to INEPT-dominant signal and a loss of birefringence in optical microscopy, suggesting an anisotropic-to-isotropic phase transition. Finally, an excellent agreement between optical microrheology and conventional mechanical rheometry was also obtained.

Live Biotherapeutic Products, A Road Map for Safety Assessment.

Recent developments in the understanding of the relationship between the microbiota and its host have provided evidence regarding the therapeutic potential of selected microorganisms to prevent or treat disease. According to Directive 2001/83/EC, in the European Union (EU), any product intended to prevent or treat disease is defined as a medicinal product and requires a marketing authorization by competent authorities prior to commercialization. Even if the pharmaceutical regulatory framework is harmonized at the EU level, obtaining marketing authorisations for medicinal products remains very challenging for Live Biotherapeutic Products (LBPs). Compared to other medicinal products currently on the market, safety assessment of LBPs represents a real challenge because of their specific characteristics and mode of action. Indeed, LBPs are not intended to reach the systemic circulation targeting distant organs, tissues, or receptors, but rather exert their effect through direct interactions with the complex native microbiota and/or the modulation of complex host-microbiota relation, indirectly leading to distant biological effects within the host. Hence, developers must rely on a thorough risk analysis, and pharmaceutical guidelines for other biological products should be taken into account in order to design relevant non-clinical and clinical development programmes. Here we aim at providing a roadmap for a risk analysis that takes into account the specificities of LBPs. We describe the different risks associated with these products and their interactions with the patient. Then, from that risk assessment, we propose solutions to design non-clinical programmes and First in Human (FIH) early clinical trials appropriate to assess LBP safety.

Simple One Pot Preparation of Chemical Hydrogels from Cellulose Dissolved in Cold LiOH/Urea.

In this work, non-derivatized cellulose pulp was dissolved in a cold alkali solution (LiOH/urea) and chemically cross-linked with methylenebisacrylamide (MBA) to form a robust hydrogel with superior water absorption properties. Different cellulose concentrations (i.e., 2, 3 and 4 wt%) and MBA/glucose molar ratios (i.e., 0.26, 0.53 and 1.05) were tested. The cellulose hydrogel cured at 60 °C for 30 min, with a MBA/glucose molar ratio of 1.05, exhibited the highest water swelling capacity absorbing ca. 220 g H2O/g dry hydrogel. Moreover, the data suggest that the cross-linking occurs via a basic Michael addition mechanism. This innovative procedure based on the direct dissolution of unmodified cellulose in LiOH/urea followed by MBA cross-linking provides a simple and fast approach to prepare chemically cross-linked non-derivatized high-molecular-weight cellulose hydrogels with superior water uptake capacity.

Facile control of surfactant lamellar phase transition and adsorption behavior.

This study sets out to investigate the effect of the presence of small water-soluble additives on the tunability of the surfactant gel-to-liquid crystalline (Lß-Lα) phase transition temperature (T m) for a bilayer-forming cationic surfactant and the phase behavior of such surfactant systems on dilution. This is strongly driven by the fact that this type of cationic surfactant has many interesting unanswered scientific questions and has found applications in various areas such as consumer care, the petrochemical industry, food science, etc. The underlying surfactant/additive interactions and the interfacial behavior of lamellar surfactant systems including the surfactant deposition on surfaces can provide new avenues to develop novel product formulations. We have examined dioctadecyldimethyl ammonium chloride (DODAC) in the presence of small polar additives, with respect to the phase behavior upon dilution and the deposition on silica. Differential scanning calorimetry (DSC) is used to track the transition temperature, T m, and synchrotron and laboratory-based small and wide-angle X-ray scattering (SAXS and WAXS) were used to determine the self-assembled surfactant structure below and above the T m. DSC scans showed that upon dilution the additives could be removed from the surfactant bilayer which in turn tuned the T m. A spontaneous transition from a liquid crystalline (Lα) phase to a gel (Lß) phase on dilution was demonstrated, which indicated that additives could be taken out from the Lα phase. By means of in situ null ellipsometry, the deposition of a diluted surfactant Lß phase upon replacement of bulk solution by deionized water was followed. This technique enables time-resolved monitoring of the deposited surfactant layer thickness and adsorbed amount, which allows us to understand the deposition on surfaces. Robust layers at least one bilayer-thick were deposited onto the surface and shown to be irreversibly adsorbed due to poor surfactant solvency in water. The thickest layer of surfactant deposited after dilution was found for mixtures with small amounts of additive since high amounts might lead to a phase-separated system.

Emulsion Formation and Stabilization by Biomolecules: The Leading Role of Cellulose.

Emulsion stabilization by native cellulose has been mainly hampered because of its insolubility in water. Chemical modification is normally needed to obtain water-soluble cellulose derivatives. These modified celluloses have been widely used for a range of applications by the food, cosmetic, pharmaceutic, paint and construction industries. In most cases, the modified celluloses are used as rheology modifiers (thickeners) or as emulsifying agents. In the last decade, the structural features of cellulose have been revisited, with particular focus on its structural anisotropy (amphiphilicity) and the molecular interactions leading to its resistance to dissolution. The amphiphilic behavior of native cellulose is evidenced by its capacity to adsorb at the interface between oil and aqueous solvent solutions, thus being capable of stabilizing emulsions. In this overview, the fundamentals of emulsion formation and stabilization by biomolecules are briefly revisited before different aspects around the emerging role of cellulose as emulsion stabilizer are addressed in detail. Particular focus is given to systems stabilized by native cellulose, either molecularly-dissolved or not (Pickering-like effect).

Exploring the prebiotic effect of cyclodextrins on probiotic bacteria entrapped in carboxymetyl cellulose-chitosan particles.

In this work the prebiotic effect of different cyclodextrins, CDs, on the viability of model probiotic bacteria (Lactobacillus rhamnosus GG) encapsulated in carboxymethyl cellulose-chitosan (CMC-Cht) hybrid particles was studied. All the CDs tested were observed to considerably improve the viability (quantitatively like common prebiotics, such as corn starch) and encapsulation efficiency when compared to the CD-free particles, as inferred by plate counting method and fluorescence microscopy. The SEM data suggests that the morphology of the particles, the roughness of the surface and porosity, are dependent on the type of CD and may reflect different interactions between the CDs and the matrix components. The aging and stability of the samples with and without ß-CD were further evaluated. Remarkably, the viability count of the CD-doped samples was still reasonably high after one month storage at room temperature with acceptable values for practical uses. Moreover, when the CMC-Cht particles were exposed to in vitro simulated digestion fluids, the cell survival was much enhanced when the particles contained ß-CD.

In-vitro digestion of curcumin loaded chitosan-coated liposomes.

Liposomes are considered a major route for encapsulation of hydrophilic and hydrophobic molecules. Chitosan coated liposomes could represent an alternative way as a carrier for delivery of drugs in human body. In this study the preparation and applicability of chitosan-coated liposomes containing curcumin as well as curcumin loaded anionic liposomes were evaluated. The applicability of the carriers was tested by means of an in-vitro digestion procedure allowing for measurement of the bioaccessibility of ingested curcumin. Values of diameter, polydispersity index and surface charge for curcumin loaded anionic liposomes obtained through dynamic light scattering and ζ-potential measurements were 129nm, 0.095 and -49mV, respectively. After chitosan-coating, diameter and polydispersity index remain unvaried while the surface charge gets positive. Slightly higher curcumin concentrations were found after the mouth and the stomach digestion phases when curcumin was loaded in anionic liposomes. On the contrary, after the intestinal phase, a higher percentage of curcumin was found when chitosan-coated liposomes were used as carrier, both in the raw digesta and in the bile salt micellar phase. It was shown that the presence of a positively charged surface allows a better absorption of curcumin in the small intestine phase, which increases the overall curcumin bioavailability. The mechanism behind these results can be understood from the composition of the different environments generated by the digestive fluids that differently interact with anionic or cationic surfaces.

On the rheology of mixed systems of hydrophobically modified polyacrylate microgels and surfactants: Role of the surfactant architecture.

HYPOTHESIS: The rheological control of suspensions is of key interest in the formulation design. A chemically cross-linked hydrophobically modified poly(acrylic acid) (HMCL-PAA), used as rheology modifier, is pH sensitive and shows swelling behavior above a critical pH due to the ionization of the acrylic acid groups. At low pH, HMCL-PAA suspensions are liquid and turbid. The binding of surfactants to HMCL-PAA, at low pH conditions, can result in significant changes on rheology and transparency of the polymeric suspensions, due to the swelling of the microgel particles. EXPERIMENTS: The influence of surfactants addition on the rheological properties and transparency of HMCL-PAA suspensions was determined. A systematic study was performed using different types of surfactants (ionic, non-ionic and zwitterionic). FINDINGS: The gelation efficiency of HMCL-PAA suspensions at low pH is strongly dependent on surfactant architecture: ionic surfactants are found to be much more efficient than non-ionic or zwitterionic surfactants. Ionic surfactants lead to a liquid-to-gel transition accompanied by an increase of transparency of the suspensions. Among the ionic surfactants, anionics show stronger interactions with the polymer. Also the surfactant hydrophobicity is relevant; the more hydrophobic the surfactant, the stronger is the binding to the polymer and thus the larger the particle swelling.

New Insights on the Role of Urea on the Dissolution and Thermally-Induced Gelation of Cellulose in Aqueous Alkali.

The gelation of cellulose in alkali solutions is quite relevant, but still a poorly understood process. Moreover, the role of certain additives, such as urea, is not consensual among the community. Therefore, in this work, an unusual set of characterization methods for cellulose solutions, such as cryo-transmission electronic microscopy (cryo-TEM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and diffusion wave spectroscopy (DWS) were employed to study the role of urea on the dissolution and gelation processes of cellulose in aqueous alkali. Cryo-TEM reveals that the addition of urea generally reduces the presence of undissolved cellulose fibrils in solution. These results are consistent with PTssNMR data, which show the reduction and in some cases the absence of crystalline portions of cellulose in solution, suggesting a pronounced positive effect of the urea on the dissolution efficiency of cellulose. Both conventional mechanical macrorheology and microrheology (DWS) indicate a significant delay of gelation induced by urea, being absent until ca. 60 °C for a system containing 5 wt % cellulose, while a system without urea gels at a lower temperature. For higher cellulose concentrations, the samples containing urea form gels even at room temperature. It is argued that since urea facilitates cellulose dissolution, the high entanglement of the cellulose chains in solution (above the critical concentration, C*) results in a strong three-dimensional network.

The relevance of structural features of cellulose and its interactions to dissolution, regeneration, gelation and plasticization phenomena.

Cellulose is the most abundant polymer and a very important renewable resource. Since cellulose cannot be shaped by melting, a major route for its use for novel materials, new chemical compounds and renewable energy must go via the solution state. Investigations during several decades have led to the identification of several solvents of notably different character. The mechanisms of dissolution in terms of intermolecular interactions have been discussed from early work but, even on fundamental aspects, conflicting and opposite views appear. In view of this, strategies for developing new solvent systems for various applications have remained obscure. There is for example a strong need for using forest products for higher value materials and for environmental and cost reasons to use water-based solvents. Several new water-based solvents have been developed recently but there is no consensus regarding the underlying mechanisms. Here we wish to address the most important mechanisms described in the literature and confront them with experimental observations. A broadened view is helpful for improving the current picture and thus cellulose derivatives and phenomena such as fiber dissolution, swelling, regeneration, plasticization and dispersion are considered. In addition to the matter of hydrogen bonding versus hydrophobic interactions, the role of ionization as well as some applications of new knowledge gained are highlighted.

Ionization of Cellobiose in Aqueous Alkali and the Mechanism of Cellulose Dissolution.

Cellulose, one of the most abundant renewable resources, is insoluble in most common solvents but dissolves in aqueous alkali under a narrow range of conditions. To elucidate the solubilization mechanism, we performed electrophoretic NMR on cellobiose, a subunit of cellulose, showing that cellobiose acts as an acid with two dissociation steps at pH 12 and 13.5. Chemical shift differences between cellobiose in NaOH and NaCl were estimated using 2D NMR and compared to DFT shift differences upon deprotonation. The dissociation steps are the deprotonation of the hemiacetal OH group and the deprotonation of one of four OH groups on the nonreducing anhydroglucose unit. MD simulations reveal that aggregation is suppressed upon charging cellulose chains in solution. Our findings strongly suggest that cellulose is to a large extent charged in concentrated aqueous alkali, a seemingly crucial factor for solubilization. This insight, overlooked in the current literature, is important for understanding cellulose dissolution and for synthesis of new sustainable materials.

From surfactant to cellulose and DNA self-assembly. A 50-year journey.

Surfactants have been the basis for applications in several industrial sectors for a long time. However, fundamental research was 50 years ago still limited to a small number of academic groups and even basic aspects were controversial. The field has since undergone an enormous expansion and the improved understanding has laid the basis of numerous new products as well as been the basis of important parts of nano-science and -technology.The present author has during 50 years in academia devoted most of his research to amphiphilic compounds, including both surfactants and polymers. Hereby, I had the privilege of following a very exciting development. In 2015, I had the honour to receive the Life-time Achievement Award of IACIS, the International Association of Colloid and Interface Scientists. IACIS organizes since the 1970s a tri-annual symposium, typically the best attended in the field. For the first time since 2000, it was in 2015 organized in Europe, namely Mainz, Germany. This treatise is based on my award lecture in Mainz, which covered developments from my first research as a new Ph D student in Stockholm to current work as an emeritus and visiting professor. Interestingly, discoveries in my very early work contributed to solving problems in now on-going research. Håkan Wennerström kindly wrote a quite comprehensive paper about my achievements a few years ago (Adv Colloid Interf Sci 205:1-8, [1]). In writing the present paper, I have strived at covering mainly topics not treated in detail by Håkan. In fact, I will emphasize very much our early studies as well as our studies of surfactant self-assembly by NMR and in particular look at the developments of our research and connections between different research topics.