Gelled Non-Toxic Microemulsions Von der Fakultät Chemie der Universität Stuttgart zur Erlangung der Würde einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung Vorgelegt von Ke Peng aus Chongqing, China Hauptberichterin: Prof. Dr. Cosima Stubenrauch Mitberichter: Prof. Dr. Thomas Hellweg Prüfungsvorsitz: Prof. Dr. Jens Brockmeyer Tag der mündlichen Prüfung: 14.10.2021 Institut für Physikalische Chemie der Universität Stuttgart 2021 Acknowledgements First and foremost, I would like to thank my supervisor Prof. Dr. Cosima Stubenrauch for offering me the opportunity to start this PhD journey. It would not have been possible without her guidance and mentoring from the very beginning till the end. I have improved so much on my research skills, scientific writing, and presentation through this journey. I am also grateful for the opportunities to attend national and international conferences and build up the network. Sincere thanks to Prof. Dr. Thomas Hellweg for being the second examiner for my thesis and Prof. Dr. Jens Brockmeyer for being the chair of the examination board. Special thanks to Prof. Dr. Thomas Sottmann for being my second supervisor and the co-author of my publications. As the expert in microemulsions and scattering techniques, he provided so much guidance and helpful advice. I am also thankful for his help and explanations with SANS measurements. I warmly thank Dr. Natascha Schelero for introducing me to the field of physical chemistry and the follow-up connections. I am also thankful to Dr. María J. Vicent for the intended collaboration; unfortunately, it did not come true due to COVID-19. Furthermore, I would like to thank Dr. Natalie Preisig for surface tension and FFEM measurements. A big thank you goes to Dr. Katja Steck for her instructions on gelation, rheology, and DLS. Sincere thanks to Kristina Schneider, Shih-Yu Tseng, and Karina Abitaev for their help with SANS measurements. Further thanks go to Dr. Sonja Dieterich for her help and Dr. Johanna Bruckner for the SAXS measurement. Sincere thanks to Birgit Feucht for her help with the visual phase studies and Diana Zauser for all the help in labs and experiments. Warm thanks to Dr. Herbert Dilger and Beate Holley for dealing with all the administration issues. Further thanks go to Gabriele Bräuning and the mechanical and electrical workshops for their support in the laboratories. Moreover, I thank all the Stubenrauch and the Sottmann group members for the shared time, scientific discussions, and hanging out. Special thanks to my office mates Friederike Dehli and Tamara Schad. Finally, thank my family for giving me the freedom to pursue my life and for all supports. Thank Douban for the mental comfort, and I am grateful to find climbing as my new hobby alongside the PhD. Sincere thanks to my dear friends for their company, especially Louise Burkart, Xiaoyue Chen, and June Li. A special and heartful thank you to Jiang. i Abstract Bicontinuous microemulsions gelled with a low molecular weight gelator (LMWG) have been found to be an orthogonally self-assembled system, i.e. the bicontinuous microstructure of the microemulsion and the fibrillar gel network form independently but simultaneously. Gelled non-toxic microemulsions have great potential to work as transdermal drug delivery systems because the non-toxic microemulsion provides optimum solubilization for drugs, while the gel network provides mechanical stability. In this work, we formulated gelled non-toxic bicontinuous microemulsions with an LMWG and investigated their formation via orthogonal self-assembly. Moreover, we provided a prototype for novel drug delivery systems. To formulate non-toxic microemulsions, we started with the scouting system H2O – n-octane – n-octyl-β-D-glucopyranoside (β-C8G1) – 1-octanol and replaced the oil and the co-surfactant with non-toxic components, namely isopropyl myristate (IPM) and 1,2-octanediol, respectively. Subsequently, the pure surfactant β-C8G1 was replaced by more efficient technical-grade sugar surfactants, including Plantacare 1200 UP (C12G1.4) and MEGA-12/14-HC (n-alkanoyl-N- methylglucamides with an average carbon chain length of 12–14). The new type of sugar surfactant, MEGA-12/14-HC, forms an ultra-efficient non-toxic microemulsion consisting of H2O – IPM – MEGA-12/14-HC – 1,2-octanediol presumably due to the electrostatic stiffening effect. However, for more reproducible results, Plantacare 1200 UP was used for further investigations. Both hydrophobic and hydrophilic model drugs, namely lidocaine and diclofenac sodium salt, were solubilized in the non-toxic microemulsion consisting of H2O – IPM – Plantacare 1200 UP – 1,2-octanediol. Moreover, phase studies were carried out at various oil-to-water ratios from ϕ = 0.2 to ϕ = 0.8. After the successful formulation of non- toxic microemulsions, the organogelator 1,3:2,4-dibenzylidene-D-sorbitol (DBS) was chosen for gelation since it does not affect the phase behavior of the non-toxic microemulsions. Rheological properties of the gelled non-toxic microemulsions were characterized by oscillatory shear rheometry. Furthermore, small-angle neutron scattering (SANS) evidenced that the gel network does not alter the microstructures of the bicontinuous microemulsion (ϕ = 0.5), and freeze-fracture electron microscopy (FFEM) images showed the coexistence of microemulsions and a fibrillar gel network. We found that an oil-continuous phase must be present for the organogelator DBS to form a strong gel and that the orthogonality of the gelled microemulsions is given for ϕ ≥ 0.35. ii Kurzzusammenfassung Bikontinuierliche Mikroemulsionen, die mit einem niedermolekularen Gelator geliert sind, haben sich als orthogonal selbstorganisiertes System erwiesen, d. h. die bikontinuierliche Mikrostruktur der Mikroemulsion und das fibrilläre Gelnetzwerk bilden sich unabhängig, jedoch gleichzeitig. Gelierte, nicht-toxische Mikroemulsionen haben dabei ein großes Potenzial für die transdermale Wirkstoffabgabe, da diese Systeme eine optimale Solubilisierung der Wirkstoffe ermöglicht, wobei das Gelnetzwerk für mechanische Stabilität sorgt. In dieser Arbeit wurden gelierte, nicht-toxische, bikontinuierliche Mikroemulsionen mit einem niedermolekularen Gelator formuliert und ihre Bildung durch orthogonale Selbstorganisation untersucht. Außerdem wurde ein Prototyp für neuartige Wirkstoffabgabesystem entwickelt. Um nicht-toxische Mikroemulsionen zu formulieren, wurde ausgehend von dem Scouting- System H2O - n-Octan - n-Octyl-β-D-glucopyranosid (β-C8G1) - 1-Octanol zunächst das Öl und das Co-Tensid durch die nicht-toxischen Komponenten Isopropylmyristat (IPM) bzw. 1,2- Octandiol ersetzt. Anschließend wurde das reine Tensid β-C8G1 durch die effizienteren technischen Zuckertenside ersetzt, nämlich Plantacare 1200 UP (C12G1.4) und MEGA-12/14- HC (n-Alkanoyl-N-Methylglucamide mit einer durchschnittlichen Kohlenstoffkettenlänge von 12–14). Das neuartige Zuckertensid MEGA-12/14-HC bildet dabei eine hocheffiziente, nicht- toxische Mikroemulsion bestehend aus H2O - IPM - MEGA-12/14-HC - 1,2-Octandiol. Die außerordentliche Effizienz ist vermutlich auf den elektrostatischen Versteifungseffekt zurückzuführen. Um jedoch reproduzierbare Ergebnisse zu erzielen, wurde für weitere Untersuchungen Plantacare 1200 UP verwendet. Sowohl ein hydrophober als auch ein hydrophiler Modellwirkstoff, nämlich Lidocain und Diclofenac-Natriumsalz, wurden in der Mikroemulsion bestehend aus H2O - IPM - Plantacare 1200 UP - 1,2-Octandiol solubilisiert. Außerdem wurde das Phasenverhalten bei verschiedenen Öl-Wasser-Verhältnissen von ϕ = 0,2 bis ϕ = 0,8 untersucht. Nach der erfolgreichen Formulierung der nicht-toxischen Mikroemulsionen wurde der Organogelator 1,3:2,4-Dibenzyliden-D-Sorbit (DBS) zum Gelieren ausgewählt, da dieser das Phasenverhalten der Mikroemulsionen nicht beeinflusst. Die rheologischen Eigenschaften der gelierten Mikroemulsionen wurden durch oszillatorische Scherrheometrie charakterisiert. Darüber hinaus zeigte die Kleinwinkel-Neutronenstreuung (SANS), dass das Gelnetzwerk die Mikrostrukturen der bikontinuierlichen Mikroemulsion (ϕ = 0,5) nicht verändert. Durch Gefrierbruch-Elektronenmikroskopiebilder (FFEM) konnte die Koexistenz von Mikroemulsionen und einem fibrillären Gelnetzwerk nachgewiesen werden. Wir fanden heraus, dass eine ölkontinuierliche Phase vorhanden sein muss, damit der Organogelator DBS ein starkes Gel bilden kann, und dass die Orthogonalität der gelierten Mikroemulsionen für ϕ ≥ 0,35 gegeben ist. iii Publication List I. Gelled Non-Toxic Microemulsions: Phase Behavior & Rheology Ke Peng, Thomas Sottmann, and Cosima Stubenrauch Soft Matter 2019, 15 (41), 8361–8371. II. Formulation of Gelled Non-Toxic Bicontinuous Microemulsions Stabilized by Highly Efficient Alkanoyl Methylglucamides Ke Peng, Natalie Preisig, Thomas Sottmann, and Cosima Stubenrauch Langmuir 2020, 36 (42), 12692–12701. III. Gelled Non-Toxic Bicontinuous Microemulsions as Promising Transdermal Drug Carriers Ke Peng, Thomas Sottmann, Cosima Stubenrauch Mol. Phys. 2021, 119 (15–16), e1886363. IV. From Water-Rich to Oil-Rich Gelled Non-Toxic Microemulsions Ke Peng, Natalie Preisig, Thomas Sottmann, and Cosima Stubenrauch Phys. Chem. Chem. Phys. 2021, 23 (31), 16855–16867. Contribution Report I. Responsible for writing the manuscript and for all experimental work except small-angle neutron scattering measurements (done by Kristina Schneider, Shih-Yu Tseng, and Diana Zauser). The co-authors revised the manuscript. II. Responsible for writing the manuscript and for most of the experimental work. The small- angle neutron scattering measurements were performed by Shih-Yu Tseng, Karina Abitaev, and Diana Zauser. Dr. Natalie Preisig carried out the freeze-fracture electron microscopy and surface tension measurements. The other co-authors revised the manuscript. III. Responsible for writing the manuscript and for all experimental work. The co-authors revised the manuscript. IV. Responsible for writing the manuscript and for all experimental work except freeze- fracture electron microscopy (done by Dr. Natalie Preisig). The co-authors revised the manuscript. iv v Contents Nomenclature ......................................................................................................................... vii 1 Introduction ................................................................................................................... 1 1.1 Motivation ....................................................................................................................... 1 1.2 Task Description ............................................................................................................. 5 2 Theoretical Background ............................................................................................... 8 2.1 Microemulsions ............................................................................................................... 8 2.2 Molecular Gels .............................................................................................................. 15 2.3 Rheology ....................................................................................................................... 20 3 Methods ........................................................................................................................ 32 3.1 Chemicals, Sample Preparation and Visual Phase Studies ........................................... 32 3.2 Oscillatory Shear Rheometry ........................................................................................ 36 3.3 Small-Angle Neutron Scattering ................................................................................... 37 3.4 Freeze-Fracture Electron Microscopy ........................................................................... 40 3.5 Electrical Conductivity ................................................................................................. 41 4 Summary of Research ................................................................................................. 42 4.1 Gelled Non-Toxic Microemulsions: Phase Behavior & Rheology (Publication I) ....... 42 4.2 Formulation of Gelled Non-Toxic Bicontinuous Microemulsions Stabilized by Highly Efficient Alkanoyl Methylglucamides (Publication II) ................................................. 49 4.3 Gelled Non-Toxic Bicontinuous Microemulsions as Promising Transdermal Drug Carriers (Publication III) ............................................................................................... 56 4.4 From Water-Rich to Oil-Rich Gelled Non-Toxic Microemulsions (Publication IV) .... 60 5 Conclusion & Outlook ................................................................................................ 68 References ............................................................................................................................... 72 vi Nomenclature vii Nomenclature Numerical 1 one-phase microemulsion 2 oil-in-water (o/w)-microemulsion (lower phase) coexisting with an excess oil phase (upper phase) 2 water-in-oil (w/o) microemulsion (upper phase) coexisting with an excess water phase (lower phase) 3 three-phase bicontinuous microemulsion coexisting with an excess water and an excess oil phase Abbreviations 12-HOA/12-HSA 12-hydroxyoctadecanoic/hydroxystearic acid 1D one-dimensional 3D three-dimensional β-C8G1 n-octyl-β-D-glucopyranoside AFM atomic force microscopy APG alkyl polyglucoside BisAm N,N’-methylenebisacrylamide C10E4 tetraethylene glycol monodecyl ether CiEj n-alkyl polyglycol ether, i is the number of carbon atoms in the n-alkyl chain, j is the number of ethylene glycol units CnGm n-alkyl polyglucoside, n is the number of carbon atoms in the n-alkyl chain, m is the number of glucose units cgc critical gelation concentration cpα the upper critical point of the binary system oil – nonionic surfactant cpβ the lower critical point of the binary system H2O – nonionic surfactant D2O deuterium oxide DBC N,N'-dibenzoyl-L-cystine DBS 1,3:2,4-dibenzylidene-D-sorbitol DBS–CONHNH2 1,3:2,4-dibenzylidene-D-sorbitol–p,p’-dihydrazide DBS–COOH 1,3:2,4-dibenzylidene-D-sorbitol–p,p’-dicarboxylic acid DLS dynamic light scattering Nomenclature viii DSC differential scanning calorimetry H2O water IPM isopropyl myristate FFEM freeze-fracture electron microscopy LMWG low molecular weight gelator LVE linear viscoelastic MEGA-n n-alkanoyl-N-methylglucamide mgc minimum gelation concentration NaCl sodium chloride NIPAm N-isopropylacrylamide NMR nuclear magnetic resonance o/w oil-in-water PIT phase inversion temperature of the microemulsion SANS small-angle neutron scattering SAXS small-angle X-ray scattering SEM scanning electron microscopy TEM transmission electron microscopy SLS static light scattering w/o water-in-oil �̃� the fishtail point on the phase diagram of a microemulsion where the 1- phase and 3-phase regions meet Parameters γ (a) surfactant mass fraction; (b) shear strain (Equation (2.7)) γ̇ shear rate (Equation (2.4) and (2.8)) γ̃ the minimal surfactant mass fraction needed to solubilize equal amounts of water and oil completely, also the composition coordinate of a microemulsion’s fishtail point, which denotes the system’s efficiency γ0 the lowest surfactant mass fraction where a microemulsion starts to form γA shear strain amplitude γC surfactant mass fraction (Equation (3.2)) γ̃C the lowest surfactant mass fraction needed to form a 1-phase microemulsion in the quaternary system H2O – oil – CnGm surfactant – co-surfactant, also one coordinate Nomenclature ix of the fishtail point γD co-surfactant mass fraction (Equation (3.3)) γ̃D the lowest co-surfactant mass fraction needed to form a 1-phase microemulsion in the quaternary system H2O – oil – CnGm surfactant – co-surfactant, also one coordinate of the fishtail point δ (a) co-surfactant mass fraction in the surfactant mixture; (b) phase shift angle (Figure 2.15) δV,i the volume fraction of co-surfactant in the interfacial surfactant monolayer ε salinity (Equation (3.4)) η (a) shear viscosity (Equation (2.5)); (b) gelator concentration (Equation (3.5)) θ scattering angle (Equation (3.6)) κ electrical conductivity λ wavelength (Equation (3.6)) ξTS correlation length of the bicontinuous microemulsion structure, a parameter in the Teubner-Strey model (Equation (3.8)) ρ scattering length density τ shear stress (Equation (3.1)) τA shear stress amplitude φ deflection angle (Equation (2.7)) ϕ oil-to-water ratio (Equation (3.1)) ω oscillation angular frequency A shear area (Equation (2.3)) c1, c2 the principal curvatures given by the two principal radii of curvature R1 and R2 of the surfactant monolayer (Equation (2.1) and (2.2)) cgelator gelator concentration csol gelator solubility in the solvent dfibril diameter of gel fibers dTS the periodicity of the oil and water domains, a parameter in the Teubner-Strey model (Equation (3.9)) F shear force (Equation (3.1)) fa the amphiphilicity factor (Equation (3.10)) G shear modulus (Equation (2.9)) G* complex shear modulus (Equation (2.17)) Nomenclature x G’ storage modulus (Equation (2.19)) G’’ loss modulus (Equation (2.20)) H the mean curvature of the interfacial surfactant monolayer (Equation (2.1)) H0 the spontaneous curvature of the interfacial surfactant monolayer ∆Hgel–sol the phase-change enthalpy of a molecular gel K the Gaussian curvature of the interfacial surfactant monolayer (Equation (2.2)) q scattering vector (Equation (3.6)) s deflection length (Equation (2.7)) t time tan δ loss factor or damping factor (Equation (2.21)) T temperature �̃� the temperature coordinate of a CiEj-type microemulsion’s fishtail point, which is also called the phase inversion temperature Tl the lowest temperature where the three-phase bicontinuous microemulsion exists Tu the highest temperature where the three-phase bicontinuous microemulsion exists Tsol–gel sol–gel transition temperature v shear velocity (Equation (2.4)) z shear gap (Equation (2.4) and (2.7)) Introduction 1 1 Introduction 1.1 Motivation Figure 1.1: Schematic illustration of orthogonal self-assembly of surfactants and low molecular weight gelators (modified from [Bri09]). Orthogonal self-assembly is the simultaneous but independent formation of two self-assembled structures. The concept was first proposed by Laibnis et al. for the simultaneous and independent formation of two self-assembled monolayers on the gold–aluminum oxide surface [Lai89]. In recent years, orthogonal self-assembly has been discovered in gelled complex fluids, which are promising for transdermal drug delivery, tissue healing, molecular electronics, or biomimetic cell membranes [Stu16]. According to the definition of Gelbert et al., a complex fluid has a mesoscopic length scale which plays a key role in determining the properties of the system [Gel96]. Examples of complex fluids are micellar solutions, microemulsions, vesicles, and liquid crystals. Complex fluids form through the self-assembly of molecules. Molecular gels form through the self-assembly of low molecular weight gelators (LMWG) [Wei14]. If the two systems are combined and if the structures form simultaneously but independently, one speaks of orthogonal self-assembly. Examples of gelled complex fluids—which are not necessarily formed via orthogonal self-assembly—are gelled micelles [Hee03], gelled vesicles Self-assembly of surfactants vesicles spherical micelles cylindrical micelles Self-assembly of low molecular weight gelators 1D-stacking Orthogonal self-assembly Introduction 2 [Bri08], gelled wormlike micelles [Bri09], gelled thermotropic liquid crystals [Kat01, Kat06, Kat07], gelled lyotropic liquid crystals [Koi17, Ste19, Ste19b, Ste19c, Ste20], and gelled liposomes [Bri09b, Boe16] (Figure 1.1). Gelled Bicontinuous Microemulsions One particularly interesting type of gelled complex fluid is a bicontinuous microemulsion gelled by an LMWG. Bicontinuous microemulsions contain two interweaving water and oil subdomains separated by an amphiphilic surfactant monolayer with domain sizes ranging from 5 to 100 nm [Str94]. The first gelled bicontinuous microemulsion was formulated by Tessendorf et al. with the ternary base system H2O – n-dodecane – Lutensol AO5 (technical- grade nonionic n-alkyl polyglycol ether with an average molecular structure of C13/15E5) and the low molecular weight organogelator 12-hydroxyoctadecanoic acid (12-HOA) [Stu07, Stu08, Mag09, Tes09]. The aim was to use the “arrested” bicontinuous microstructure as a template for the synthesis of bicontinuous (sponge-like) nanoporous polymers. Therefore, the polymerizable aqueous phase contained the monomer N-isopropylacrylamide (NIPAm) and the cross-linker N,N’-methylenebisacrylamide (BisAm). Freeze-fracture electron microscopy (FFEM) images and small-angle neutron scattering (SANS) data of the non-polymerized systems suggested that the bicontinuous microstructure was not altered by the gel network. In other words, the gelled bicontinuous microemulsion was considered to be an orthogonal self- assembled system, where the bicontinuous microemulsion and the fibrillar gel network form simultaneously but independently. Further investigations on gelled bicontinuous microemulsions were carried out by Laupheimer et al. with the model system H2O – n-decane / 12-HOA – tetraethylene glycol monodecyl ether (C10E4) [Lau13, Lau13b, Lau14]. The characteristic properties and microstructures of the gelled bicontinuous microemulsion were compared with those of the two base systems, namely the non-gelled bicontinuous microemulsion H2O – n-decane – C10E4 and the binary gel n- decane / 12-HOA. The phase boundaries of the non-gelled microemulsion were maintained Introduction 3 upon gelation but shifted to lower temperatures because 12-HOA is surface-active. The gelled microemulsion and the binary gel had similar rheological properties. The mere differences are that the gel network in the gelled microemulsion is slightly weaker than in the binary gel, and the sol–gel transition temperature of the gelled microemulsion is lower than that of the binary gel. These differences can be explained by the fact that (a) the surface-active 12-HOA molecules partly adsorb at the water–oil interface instead of forming gel fibers, (b) the change of the gel’s solvent from pure n-decane to a microemulsion leads to different gelator–solvent interaction and thus to slightly different rheological properties. Moreover, the coexistence of two self-assembled structures—the fibrillar gel network and the bicontinuous microstructure— was evidenced with the help of FFEM and SANS. Opposed to the assumption of Tessendorf that gel fibers only exist in the oil domain since 12-HOA is an organogelator, the gel fibers pass through the entire microemulsion, i.e. through both water and oil domains. Non-Toxic Microemulsions Non-toxic microemulsions are of great interest for drug delivery [Kre02, Heu08, Law12]. However, most of the microemulsions used in drug delivery are oil-in-water (o/w) type microemulsions, while bicontinuous microemulsions are hardly used [Cal17]. This is remarkable since bicontinuous microemulsions provide the optimum solubilizing capacity for drugs and facilitate the drug permeation through the skin barrier [Bol98, Bol99]. Several drug- containing bicontinuous microemulsions have been formulated up to now. (1) A dermal delivery system consisting of H2O – isopropyl myristate (IPM) – polysorbates containing the local anesthetic lidocaine was characterized by NMR self-diffusion. However, the system was water-rich, and the surfactant mass fraction was as high as 30 wt.%, which means that a bicontinuous structure is not very likely [Car91]. (2) Froelich et al. presented bicontinuous microemulsions consisting of H2O – oleic acid – polysorbates using ethanol and diethylene glycol monoethyl ether as co-surfactants [Fro17]. However, the total amount of surfactant and co-surfactant was as high as 70 wt.%, and only surface tension and electrical conductivity were measured, while no structural characterization was carried out. (3) In another bicontinuous Introduction 4 microemulsion, an alkyl ester was used as oil, sucrose esters as surfactant, and diethylene glycol monoethyl ether as co-surfactant [Bol98, Bol99]. The bicontinuous microstructure was evidenced by freeze-fracture electron microscopy (FFEM) and small-angle neutron scattering (SANS). However, the system was only studied on the oil-rich side, and the surfactant content was quite high (18 wt.% surfactant and 12 wt.% co-surfactant). Note that high surfactant contents are not preferred due to toxicity and economical considerations. Kahlweit et al. made a series of attempts to formulate non-toxic microemulsions and carefully studied the phase behavior for the purpose of potential pharmaceutical applications. They started with the quaternary system H2O – n-alkane – lecithin – alkanol with equal weights of water and oil [Kah95a] and replaced the zwitterionic surfactant lecithin with nonionic alkyl polyglucosides (APGs, or glycosides) [Kah95b]. It turned out that APGs are efficient enough to form bicontinuous microemulsions, which was important progress because technical-grade APGs are less expensive than lecithin. Since the mineral n-alkanes are not biocompatible, Kahlweit et al. replaced n-alkanes with unsaturated fatty acid ethyl esters and replaced alkanols with alkane-1,2-diols, which are biocompatible yet surface-active enough to act as co- surfactant [Kah95c, Kah96]. Finally, due to its sensitivity to light and oxygen, the unsaturated fatty acid ethyl ester was replaced by the more stable saturated fatty acid ester isopropyl myristate (IPM). In this way, a non-toxic bicontinuous microemulsion consisting of H2O – IPM – APG – 1,2-octanediol was successfully formulated [Kah97]. Gelled Non-Toxic Microemulsions Gelled non-toxic microemulsions are preferred to their non-gelled counterparts in transdermal drug delivery due to the mechanical stability, which provides an easy application and immobilizes drugs on the applied surface [Law12]. So far, most of the gelled microemulsions are polymer gels [Kan99, Cru01, Xua12, Fou13, Fro17], whose gel networks form through chemical and/or physical cross-linking of polymeric chains. In contrast, LMWG molecules self-assemble into gel networks through non-covalent interactions (physical). In the thesis at Introduction 5 hand, the goal is to combine the novel concept of forming gelled bicontinuous microemulsions via orthogonal self-assembly with the formulation of non-toxic microemulsions and explore their potentials in transdermal drug delivery (Figure 1.2). Molecular gel networkNon-toxic microemulsion Gelled non-toxic microemulsion low molecular weight gelator hydrophobic drug hydrophilic drug Figure 1.2: Schematic presentation of a drug-loaded gelled non-toxic microemulsion. 1.2 Task Description In order to study the formation of gelled non-toxic microemulsions via orthogonal self- assembly and their potential use as a drug delivery system, three tasks need to be dealt with: (1) Formulation of non-toxic microemulsions with biocompatible components and model drugs. (2) Identification of a suitable low molecular weight gelator (LMWG) for the gelation of the microemulsion. (3) Examination of whether the orthogonality of the two structures is maintained, i.e. whether the system is formed via orthogonal self-assembly of the surfactant and the gelator. (1) Regarding the formulation of non-toxic microemulsions, one first needs to find a biocompatible surfactant. Sugar surfactants, such as n-alkanoyl-N-methylglucamides (MEGA-n, Figure 1.3) and alkyl polyglucosides (APGs, denoted as CnGm, an example of CnG1 is given in Figure 1.3), are dermatologically safe, commercially available, and exhibit excellent surface activity [Hil99]. Due to their hydrophilicity, sugar surfactants form bicontinuous microemulsions only with the addition of co-surfactants. The APG-based model system H2O – n-octane – n-octyl-β-D-glucopyranoside (β-C8G1) – 1-octanol was extensively studied with Introduction 6 respect to its phase behavior, interfacial composition, interfacial tension, and microstructure [Klu00, Klu01, Sot02, Rei03]. As the oil n-octane and the co-surfactant 1-octanol are irritating to human bodies, they must be replaced by non-toxic saturated fatty acid esters, such as isopropyl myristate (IPM, Figure 1.3) and alkanediols (one example is given in Figure 1.3) [Kah96, Kah97]. Both IPM and alkanediols are bio-compatible and act as permeation enhancers in transdermal drug delivery [Kog06, Joh12, Wil12]. Based on the quoted previous studies, the tasks of the present thesis are the following: (a) Restudy the phase behavior of the scouting system H2O – n-octane – β-C8G1 – 1-octanol at T = 25 °C with equal volumes of water and oil to ensure a bicontinuous structure. (b) Replace the oil n-octane and the co-surfactant 1-octanol of the scouting system with non-toxic components, namely the oil IPM and the co-surfactant 1,2-octanediol. (c) Search for a more efficient and less expensive sugar surfactant, e.g. technical-grade APGs with longer carbon chains. (d) Formulate model drugs in the non-toxic bicontinuous microemulsions, namely the hydrophobic model drug lidocaine and the hydrophilic model drug diclofenac sodium salt (Figure 1.3). (e) Vary the oil-to-water ratios systematically along the trajectory of the middle phase to extend the application potential of the gelled non-toxic microemulsions. n-alkyl-β-D-glucopyranoside (β-CnG1)n-alkanoyl-N-methylglucamides (MEGA-n) isopropyl myristate (IPM) 1,2-octanediol lidocaine diclofenac sodium salt Figure 1.3: Molecular structures of used chemicals. Introduction 7 (2) Since the bicontinuous microemulsion contains equal volumes of oil and water, theoretically, both organogelator and hydrogelator could be suitable to gel the microemulsion. The chosen candidates are organogelators 12-hydroxyoctadecanoic acid (12-HOA) and 1,3:2,4- dibenzylidene-D-sorbitol (DBS), and hydrogelators N,N'-dibenzoyl-L-cystine (DBC) and DBS derivatives, namely 1,3:2,4-dibenzylidene-D-sorbitol–p,p’-dicarboxylic acid (DBS–COOH) and DBS–CONHNH2 (Figure 2.8). 12-HOA is surface-active [Lau13], which thus might act as both gelator and co-surfactant, and ideally no additional co-surfactant other than 12-HOA would be needed for the formation of a microemulsion. In contrast, DBS and DBC are not surface-active, and the phase boundaries of gelled microemulsions should be consistent with those of the non-gelled counterparts. These gelators are first to be tested in the scouting system and then in the non-toxic system. With the variation of the oil-to-water ratios of the non-toxic microemulsion, two questions are to be answered: (a) Does the same gelator gel both water- rich and oil-rich microemulsions? (b) If not, does a hydrogelator gel the water-rich microemulsion and an organogelator the oil-rich microemulsion? The gel properties of the gelled non-toxic microemulsions are to be characterized by oscillation shear rheometry. The storage modulus G’ and the loss modulus G’’ are to be measured by frequency sweeps and the sol–gel transition temperature Tsol–gel by temperature sweeps. (3) In order to characterize the orthogonality of the gelled non-toxic microemulsion, the gelled non-toxic microemulsion is to be compared with the non-gelled counterpart. Firstly, the phase diagrams of both systems are to be compared to see whether the gelator affects the phase behavior of the bicontinuous microemulsion. Secondly, small-angle neutron scattering (SANS) is to be used to characterize the microstructure of the two systems. Thirdly, freeze-fracture electron microscopy (FFEM) is to be used to (i) visualize the coexistence of the bicontinuous microemulsion and the fibrillar gel network, and (ii) confirm the length scales of the microemulsion domains and of the gel network. Theoretical Background 8 2 Theoretical Background In the following sections, the two base systems of gelled non-toxic microemulsions are introduced, namely microemulsions and molecular gels. If not otherwise stated, the theory of microemulsions was taken from [Sot05, Sot08] and that of molecular gels from [Wei06, Geo06, Zwe13]. Additionally, the theoretical principles of rheology are introduced, of which the theory was taken from [Mez06, Bla18]. 2.1 Microemulsions A microemulsion consists of at least three components, namely a hydrophilic, a hydrophobic, and an amphiphilic one. In most cases, these three components are water, oil, and surfactant. Microemulsions have two characteristics: they are thermodynamically stable, and they have various nanostructures, which distinguish them from emulsions. The domain size of microemulsions is around 3–100 nm [Str94], whereas the droplet size of emulsions is within the μm range. Phase Behavior Phase behavior studies of microemulsions are necessary to determine the conditions where the surfactant solubilizes the maximum amounts of water and oil. The ternary systems consist of water, oil, and the nonionic surfactant n-alkyl polyglycol ethers (CiEj, where i denotes the hydrophobic carbon chain length, and j denotes the number of ethylene oxide groups) [Kah85]. These ternary nonionic microemulsions have been extensively studied [Kah87], and they are taken as an example to illustrate the general phase behavior of microemulsions. To understand the complex behavior of the ternary system, one first needs to understand the binary base systems. Figure 2.1 depicts phase diagrams of the three binary systems. The binary system water – oil has a large miscibility gap. In the binary system oil – nonionic surfactant, the upper critical point cpα is typically located below 0 °C, which means that the nonionic Theoretical Background 9 surfactant is completely soluble in oil under normal experimental conditions. The most interesting binary phase diagram is the one of water – surfactant [Str96], where both an upper and a lower miscibility gap exist. Since the lower miscibility gap is far below 0 °C (not shown in Figure 2.1), it is of no relevance for the following discussion. The upper miscibility gap has a lower critical point cpβ. At high concentrations of surfactants, lyotropic liquid crystals can form (not shown in Figure 2.1). What is important for understanding the phase behavior of microemulsions stabilized with CiEj surfactants is the fact that the solubility of CiEj surfactants in water decreases with increasing temperature, whereas their solubility in oil increases with increasing temperature. These trends are due to an increase in hydrophobicity as a result of the dehydration of the head groups. H2O (A) oil (B) nonionic surfactant (C) T T T 0 °C 0 °C Figure 2.1: Unfolded phase prism of the ternary system H2O – oil – nonionic surfactant with the miscibility gaps of the three binary systems (redrawn from [Sot08]). Folding up the three binary systems, one obtains a complex ternary phase prism [Kah87]. Since one major tuning parameter for the phase behavior is the temperature T, a simplified method to study the phase behavior is to measure a so-called “fish-cut”, i.e., a T(γ)-section through the phase prism at a constant oil-to-water ratio ϕ (Figure 2.2). Here γ denotes the total mass fraction of the surfactant in the sample. Below γ0 the surfactant concentration is too low to form a microemulsion. Between γ0 and γ̃ water and oil are only partially solubilized, and hence excess water and oil phases form. With increasing temperature, phase transitions occur in the sequence Theoretical Background 10 of 2 − 3 − 2. Below Tl, the sample consists of an o/w-microemulsion (lower phase) and an oil excess phase (upper phase), denoted as “2” since the surfactant-rich phase is the lower phase. Between Tl and Tu, a bicontinuous microemulsion middle phase coexists with a water (lower phase) and an oil excess (upper phase) phase, denoted as “3”. Above Tu, the sample consists of a water excess phase (lower phase) and a w/o-microemulsion (upper phase), accordingly denoted as “2” because the surfactant-rich phase is the upper phase. At a γ higher than γ̃, 2 and 2 still exist, but the three-phase region transforms into an isotropic bicontinuous phase without excess oil and water (denoted as “1”). The fishtail point �̃� has the coordinates γ̃ and �̃�. γ̃ represents the minimal mass fraction of surfactant needed to solubilize equal amounts of water and oil completely, which is a measure of the surfactant efficiency. �̃� is called phase inversion temperature (PIT). γ 𝑇 T 13 2 2 �̃� ̃ �̃� 𝑇 ϕ = 0.5 Figure 2.2: Schematic T(γ)-section (“fish-cut”) at a constant oil/(oil + water) volume fraction ϕ = 0.5. The test tubes illustrate the relative volumes of phases. Another type of nonionic surfactant is sugar surfactants, whose phase behavior differs from that of CiEj type surfactants. Sugar surfactants are of great interest because of their nontoxicity and biodegradability. They are widely used in personal care, pharmaceutics, and detergents [Hil99], and they can be manufactured from renewable resources. Sugar surfactants consist of a hydrophobic alkyl chain and a hydrophilic sugar head group. The bond between the alkyl Theoretical Background 11 chain and the sugar head group can be an ester, an ether, an amine, or an amide. Alkyl glycosides are the most extensively studied sugar surfactants because of their excellent surface activities. They are often denoted as CnGm, where n denotes the alkyl chain length and m denotes the number of glucose units. Figure 2.3: (Left) A schematic phase tetrahedron of quaternary system H2O – oil – surfactant – co-surfactant at a constant temperature (modified from [Klu01]). (Right) A schematic section through the phase tetrahedron of the quaternary system H2O – oil – CnGm surfactant – co- surfactant at ϕ = 0.5 and a constant T. One characteristic property of CnGm is that microemulsions stabilized with CnGm surfactants are less temperature-sensitive than those stabilized with CiEj surfactants. This is due to the strong hydrogen bonds between hydroxyl groups and water molecules, making dehydration difficult, even at high temperatures [Stu01]. Due to the temperature insensitivity, the phase behavior cannot be tuned by changes of temperature. The sugar head group is so hydrophilic that in the ternary system H2O – oil – CnGm only o/w-microemulsions form. Therefore, a relatively hydrophobic co-surfactant (such as long-chain alcohol) is necessary to reach the middle point of a fish-cut, i.e. to form a bicontinuous microemulsion. In contrast to the phase prism of the ternary system of H2O – oil – CiEj surfactant, the phase behavior of the quaternary system of H2O – oil – CnGm surfactant – co-surfactant is depicted in an isothermal phase tetrahedron (Figure 2.3 (left)), which consists of four Gibbs phase triangles of the corresponding ternary systems. At a constant oil-to-water ratio ϕ, a γD(γC)-section through the phase tetrahedron can be obtained, (ϕ = 0.5 as an example in Figure 2.3 (right)). The two axes ϕ = 0.5 C D ϕ = 0.5 1 3 2 2 �̃� ̃C ̃D Theoretical Background 12 present the mass fractions of the surfactant (C) and the co-surfactant (D), γC and γD, respectively. The fishtail point �̃� has the coordinates γ̃C and γ̃D . When γC is smaller than γ̃C , phase transitions occur in the sequence of 2 − 3 − 2 along with the addition of a co-surfactant. When γC is larger than γ̃C, the increase of γD induces the phase transition sequence of 2 − 1 − 2. At high surfactant concentrations, the lamellar phase exists in the 1-phase region. Microstructure Microemulsions are macroscopically homogeneous but heterogeneous on the nanoscopic scale. The main parameter determining the microstructure is the mean curvature H of the interfacial surfactant monolayer, which is defined as 𝐻 = 𝑐1 + 𝑐2 2 (2.1) with 𝑐1 = 1 𝑅1⁄ and 𝑐2 = 1 𝑅2⁄ being the principal curvatures given by the two principal radii of curvature R1 and R2 of the surfactant monolayer. The curvatures are positive if the surfactant monolayer bends towards oil (o/w-microemulsion) and negative if it bends towards water (w/o-microemulsion). The mean curvature H, which can be experimentally determined [Str94], is closely related to the spontaneous curvature H0, i.e. the curvature of the interfacial surfactant monolayer if no external forces, thermal fluctuations, or conservation constraints exists. Another important parameter is the Gaussian curvature K, which is given by 𝐾 = 𝑐1𝑐2. (2.2) The variation of the mean curvature H of the interfacial surfactant monolayer can be induced by the change of temperature T, co-surfactant ratio δ, or salinity, etc. Figure 2.4 (left) illustrates the variation of the mean curvature H for the temperature-sensitive ternary system of H2O – oil – CiEj. At low temperatures, the ethylene oxide head groups of CiEj surfactants are strongly hydrated, so the volume of the hydrophilic head groups in water is larger than the volume of the hydrophobic tails in oil, leading to a positive mean curvature. Thus, the surfactant Theoretical Background 13 monolayer tends to enclose water, and o/w-microemulsions form. With increasing temperatures, the ethylene oxide head groups become less and less hydrated, which decreases the volume of the head groups, while the volume of the hydrophobic tails remains the same, resulting in a lower mean curvature H. When H = 0, the microstructure of the system transforms from discontinuous droplets in a continuous phase into two continuous phases. As the temperature increases further, the head groups become completely dehydrated so that H eventually turns into negative values. The surfactant monolayer tends to enclose oil, and w/o-microemulsions form. The same principle applies to the less temperature-sensitive quaternary system consisting of H2O – oil – CnGm – co-surfactant (Figure 2.4 (right)). The strong hydration of the sugar surfactant leads to large head groups and thus to a positive mean curvature H. The more hydrophobic co-surfactant has a smaller head group. Therefore, the addition of co-surfactants leads to the incorporation of the co-surfactant molecules into the surfactant monolayer, which causes a decrease of the mean curvature from H > 0 to H < 0. Here, the composition of the interfacial surfactant monolayer δV,i [Sot02] is the tuning parameter of the mean curvature. Figure 2.4: Mean curvature H of a nonionic surfactant monolayer at the water–oil interface as a function of (left) temperature T and (right) composition of the interfacial surfactant monolayer δV,i. Various experimental methods are available to characterize the microstructure quantitatively. Transmission electron microscopy provides direct and local information on the nanoscale [Jah88, Bur03]. Small-angle X-ray/neutron scattering (SAXS/SANS) determines the statistical length scales [Hel08]. NMR self-diffusion [Lin96] and electrical conductivity measurements H > 0 H = 0 H < 0 T H > 0 H = 0 H < 0 Theoretical Background 14 [Kah87] provide valuable information on the connectivity and on structural transitions. With the help of these experimental methods, extensive microstructure studies bring together an overview of the microstructure transitions caused by the change of the temperature and the surfactant concentration in the ternary systems H2O – oil – CiEj (Figure 2.5). At the phase inversion temperature �̃�, the microemulsion always has a mean curvature of H = 0. At low surfactant concentrations near the �̃�-point, bicontinuous structure exists in the middle phase of the 3-phase region and in the 1-phase region. The bicontinuous structure consists of two continuous sub-phases – water and oil – which are separated by a surfactant monolayer. The two domains interweave with each other and form a sponge-like structure, which has a mean curvature of H = 0 but a Gaussian curvature of K < 0 because c1 and c2 have opposite signs. As the surfactant concentration increases, the structure length scale becomes smaller because the total area of the internal interface increases. At high surfactant concentrations, the lamellar phase Lα is observed with a zero-curvature structure, being H = 0 and K = 0 (c1 = c2 = 0). Moving away from the �̃�-point, γ > γ̃, o/w- or w/o-droplets appear at low or high temperatures, respectively. The droplet size decreases as γ further increases. During the transition from the bicontinuous structure to the droplet structure, one also observes elongated cylindrical domains. Figure 2.5: Schematic overview of the microstructures induced by the variation of temperature and surfactant concentration in nonionic microemulsions consisting of H2O – oil – CiEj. The white regions represent water, and the shaded regions represent oil. Modified from [Str94]. 2 2 3 �̃� ̃ T γ oil in water water in oil �̃� Theoretical Background 15 2.2 Molecular Gels Definition and Characteristics Gels have been discovered for more than 150 years [Gra61]. Over the years, the definition of gels continues evolving. Generally, a substance is regarded as a gel if it (i) has a continuous microscopic structure that is permanent on the time scale of analytical experiments and (ii) has solid-like rheological behavior despite consisting mostly of a fluid. A gaseous or liquid system consisting of at least two components forms a gel if one of the components forms a three- dimensional (3D) entangled solid network within the bulk gas or liquid phase. The solid network restricts the flow of the remaining fluid bulk phase, which macroscopically appears as a solid. There are several possibilities to classify gels, e.g., according to the physical state of the bulk phase or the chemical nature of the components. A practical classification is to distinguish between chemical and physical gels. In chemical gels, e.g. cross-linked polymeric systems, the formation of a 3D network occurs through covalent cross-linking of polymers. The network structure is permanent unless the covalent cross-links are broken. In physical gels, the formation of a 3D network occurs through non-covalent interactions, such as hydrogen bonds, van der Waals forces, dipolar interactions, and π–π stackings. Thus, the formation of physical gels is thermoreversible. Physical gels can be formed with various substances, e.g. clays, polymers, proteins, colloids, and certain small organic compounds. Molecular gels are physical gels formed by small organic compounds called low molecular weight gelators (LMWGs). LMWG molecules have a molecular weight of less than 2000 Da, and they can gel organic solvents or water. If an LMWG gels organic solvents, it is called an organogelator. If an LMWG gels water, it is called a hydrogelator. Theoretical Background 16 Figure 2.6: Schematic depiction of the molecular gel formation process. The formation process of molecular gels is schematically depicted in Figure 2.6. Molecular gels are most commonly prepared by cooling a solution (or sol) with a low concentration of an LMWG (frequently ≤ 2 wt.%) in the solvent to be gelled below the characteristic gelation temperature. Thus, the solution is supersaturated, and microscopic phase separation occurs, which leads to the crystallization of LMWG molecules. The LMWG molecules self-assemble in stochastic nucleation events, and highly specific non-covalent interactions allow preferential one-dimensional nuclei growth into crystal-like fibers. These fibers have the same function as polymers in polymer gels and aggregate into a fibrous 3D entangled network. The essential formation of so-called “junction zones” (nodes) [Ter95] provides rigidity to the 3D network and distinguishes it from an aggregate of 1D fibrillar objects which do not interact. The shapes of junction zones range from strands, tapes, chiral ribbons, and tubules to other various aggregates with large aspect ratios. Note that the aggregation of gelator molecules and gel fibers occurs through non-covalent interactions, which are considerably weaker than covalent bonds. Two important characteristics of LMWGs are the minimum gelation concentration (mgc) and the gel–sol phase transition temperature Tsol–gel. The minimum gelation concentration, also called the critical gelation concentration (cgc), is the minimum amount of the gelator to gel a specific solvent. The gel–sol phase transition temperature Tsol–gel is the temperature at which the gel loses its structural integrity. Tsol–gel depends on the molecular structure of the gelator, the nature of the solvent, and the total gelator concentration. With the determination of Tsol–gel in a solvent over a range of gelator concentrations, one can establish a phase diagram for the freely dissolved LMWG molecules in solution LMWG molecules self- assemble into fibers 3D entangled fibrillar network Theoretical Background 17 gelator–solvent combination (Figure 2.7). The sol phase is the solution of gelators in the solvent, while the gel phase consists of the gelator solution and the 3D gel network (solid). Thus, the gel-to-sol transition line is a measure of the gelator solubility in the solvent csol [Chr18]. As the gelator concentration increases, Tsol–gel also increases until it reaches a plateau. Above the gelator concentration threshold, where the Tsol–gel reaches a plateau, the melted gelator and the solvent are no longer miscible, corresponding to a monotectic transformation [Chr16]. Figure 2.7: Example of a phase diagram for a gelator in a solvent. Versatile experimental techniques are available to determine phase diagrams and to characterize the macroscopic behavior of molecular gels. The “tabletop” rheology can be carried out in a laboratory without sophisticated equipment, which gives a “quick and dirty” overview of the general properties such as mgc and Tsol–gel. One can thus quickly establish a phase diagram of a specific solvent–gelator system. The most common methods are tube inversion and dropping ball [Rag06]. A more precise yet simple method to map the phase diagram is liquid NMR, which yields the gelator solubility as a function of temperature with a single sample [Chr18]. Quantitative characterizations of the viscoelastic properties can be obtained by oscillatory rheology (see Section 2.3 for more details). Depending on the measuring programs, oscillatory rheology characterizes the yield point, the gel–sol transition temperature Tsol–gel, and the recovery time after destructive stress (thixotropy). Differential scanning calorimetry (DSC) determines the gel–sol transition temperature (both on heating and cooling) and the phase-change enthalpy (∆Hgel–sol), which provides insight into the thermodynamics of intermolecular actions [Rag06]. On the nanoscale, the fiber and gel 𝑐 sol (= solution) gel (= solution + solid) 𝑇 𝑇 𝑐 Theoretical Background 18 morphology can be visualized with transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additionally, atomic force microscopy (AFM) can provide information on fiber width and height [Neb13, Gue18]. Instead of the bundled/aggregated gel fibers observed in electron microscopy images, small-angle X-ray/neutron scattering (SAXS/SANS) provides averaged information about the dimensions of molecular fibrils [Ter06]. Low molecular weight gelators (LMWGs) In the thesis at hand, five LMWGs with a wide variety of molecular structures were chosen (see Figure 2.8 for molecular structures). We chose two organogelators, namely 12- hydroxyoctadecanoic acid (12-HOA) and 1,3:2,4-dibenzylidene-D-sorbitol (DBS). In addition, we studied three hydrogelators, namely N,N'-dibenzoyl-L-cystine (DBC) and the derivatives of DBS, 1,3:2,4-dibenzylidene-D-sorbitol–p,p’-dicarboxylic acid (DBS–COOH) and 1,3:2,4- dibenzylidene-D-sorbitol–p,p’-dihydrazide (DBS–CONHNH2). The gelation demands a meticulous balance between solubility and crystallinity of LMWGs. If the gelator is highly soluble in the solvent, gelation does not occur. If the gelator is too insoluble, it precipitates or crystallizes rapidly. Therefore, although the chemical properties and the molecular structures of the LMWGs do play a role, it remains trial and error to know which gelator gels which solvent. In the following, the properties of the five gelators will be briefly described. Figure 2.8: Molecular structures of the chosen LMWGs. 1,3:2,4-dibenzylidene-D-sorbitol (DBS, R = H) and its derivatives N,N'-dibenzoyl-L-cystine (DBC) 12-hydroxyoctadecanoic acid (12-HOA) Theoretical Background 19 12-Hydroxyoctadecanoic/hydroxystearic acid (12-HOA/12-HSA) is one of the most widely studied LMWGs, which belongs to the long-chain saturated fatty acid type. The unique self- assembly behavior of 12-HOA originates from its chemical structures of a carboxylic head group and a secondary hydroxyl group. The presence of a hydroxyl group at position 12 on the C18-fatty acid chain induces interaction possibilities and molecular chirality. Hence, 12-HOA can gel various organic solvents, such as vegetable oils, long-chain alkanes, cycloalkanes, long- chain aldehydes, ethers, short-chain alkanediols, nitriles, and thiols [Gao12, Lan15]. The intermolecular interactions of 12-HOA are mainly hydrogen bonding. The carboxylic acid head group of the molecule forms cyclic dimers with the adjacent molecule, meanwhile facilitating interactions between hydroxyl groups. Both interactions lead to unidirectional hydrogen bonding sequences along the fiber axis [Ter94, Sat08, Sat11]. Furthermore, 12-HOA with alkali metals or alkanolamines as counterions also gels aqueous solutions [Fam20]. Various structures are reported, such as fibers, tubes, and helical ribbons [Lau15]. The structures depend on the enantiomer purity of 12-HOA, the type of counterions, and the molar ratio between 12-HOA and counterions. DBC as an amino acid derivative is an efficient hydrogelator. It gels water at a very low gelator content of 0.1 wt.% (2 mM) and forms a firm, translucent gel at a concentration of 4 mM in ethanol (5%)/water. The self-assembly of DBC is caused by hydrogen bonds between amide- NH and carboxyl-CO functionalities. Besides, π–π stacking of the aromatic rings promotes the formation of gel fibers. Furthermore, the disulfide linkage –S–S– is critical for the gelation, presumably because the disulfide linkage forms a linear backbone, and its conformational rigidity favors hydrogen-bonding contacts. Overall, gel fibers are held together by a favorable backbone orientation enhanced by hydrogen bonding and π–π stacking or hydrophobic interactions [Men95, Men00]. Note that the gelation of DBC only occurs when the pH is below 5 [Zie13]. At basic conditions, the ionized carboxyl groups destroy the gel due to electrostatic repulsion. Theoretical Background 20 Figure 2.9: A conformational perspective of the molecular structure of 1,3:2,4-dibenzylidene- D-sorbitol (DBS, taken from [Oke15]). DBS is a well-known organogelator of the sorbitol derivative type. It has been widely used in cosmetics, biomedical materials, and electronic devices. DBS has a chiral butterfly-shaped conformation – the D-sorbitol backbone is the “body,” and the phenyl rings are the “wings”. The butterfly-like DBS has two potential gelation mechanisms: (1) hydrogen bonding of the “body” with 5-OH/6-OH groups (see Figure 2.9) of one molecule being hydrogen bond donor and the 5-OH/6-OH or the cyclic acetals of another being hydrogen bond acceptor, and (2) π– π stacking or solvophobic interactions between the aromatic “wings” of two molecules. Experimental and theoretical results reveal that (a) in non-polar solvents, intermolecular hydrogen bonding, mainly from the 6-OH group, plays a key role in self-assembly; (b) in polar or protic solvents, intermolecular hydrogen bonding becomes less significant, while π–π stacking or solvophobic interactions between the aromatic “wings” become more dominant [Oke15]. To increase the solubility of DBS in water, one can modify DBS with additional functional groups on the aromatic rings. The resulting DBS derivatives can act as hydrogelators. DBS–COOH requires protonation of the carboxyl groups to gel. Thus the gelation is induced by slow acidification of DBS–COOH in a basic solution [Cor13]. The other hydrogelator DBS– CONHNH2 is pH-tolerant, which is potentially easier to use than DBS–COOH [Oke13]. 2.3 Rheology Rheology is essential to characterize quantitatively the mechanical properties of gels. Rheological measurements provide information about the flow behavior of liquids and the deformation behavior of solids caused by shear forces. All types of rheological behavior are Theoretical Background 21 between the two extreme cases: the flow behavior of ideally viscous liquids and the deformation behavior of ideally elastic solids. Viscous behavior The Two-Plates-Model is used to define the fundamental rheological parameters (Figure 2.10 (left)). An upper plate with a shear area A is set in motion by the shear force F, resulting in a velocity v to be measured. The distance between the upper plate and the stationary lower plate (v = 0) is the shear gap z. The sample is sheared in this shear gap. If there are no wall-slip effects, the sample adheres to both plates, and if the flow is laminar, the shear stress is defined as τ = 𝐹 𝐴⁄ [Pa]. (2.3) The shear rate is defined as γ̇ = 𝑑𝑣 𝑑𝑧⁄ = 𝑣 𝑧⁄ [s-1], (2.4) given laminar flow conditions (dv = const., dz = const.). Accordingly, the shear viscosity is given by η = τ γ̇⁄ [Pa·s]. (2.5) Figure 2.10: Schematic presentations of (left) the Two-Plates-Model for flow behavior, (right) the dashpot model. vA z F v = 0 v(z) Theoretical Background 22 τ γ̇ a b c η γ̇ a b c Figure 2.11: Flow curves (left) and viscosity functions (right) of the three types of flow behaviors: a = ideally viscous, b = shear-thinning, c = shear-thickening. The ideally viscous (or Newtonian) flow behavior fulfills Newton’s law τ(𝑡) = η ∙ γ̇(𝑡), (2.6) i.e., the τ(γ̇)-flow curve is a straight line and has a constant slope (Figure 2.11 (left)). Analogously, the viscosity function η(γ̇)-curve of an ideally viscous or Newtonian fluid is a flat line (Figure 2.11 (right)) since the shear viscosity is independent of the magnitude and duration of the shear load applied. The flow behavior of a Newtonian fluid can be represented by the dashpot model (Figure 2.10 (right)). Under a constant force, the piston and the dashpot fluid move with a constant velocity or deformation rate, and the velocity is proportional to the force strength. The proportionality corresponds to the internal friction of the dashpot, i.e. the viscosity. Once the force is removed, the piston stops moving immediately, and thus the ideally viscous fluid remains deformed. During the flow or deformation process, the applied deformation energy is converted to frictional heating (also called viscous heating) between molecules. The resulting thermal energy partially heats up the fluid and partially goes to the surrounding environment. Therefore, the deformation process is irreversible. In ideally viscous fluids, such as water, oils, and pure solvents, there are no strong interactions between molecules. However, there are also other types of fluids, namely shear-thinning and shear-thickening fluids (Figure 2.11). For shear-thinning fluids, the viscosity decreases with increasing shear rate, and the slope of the τ(γ̇)-flow curve decreases with increasing γ̇. Vice versa, for shear-thickening fluids, the viscosity increases with increasing shear rate, and the slope of the τ(γ̇)-flow curve increases. Theoretical Background 23 Elastic Behavior The Two-Plates-Model can also be used to define the elastic behavior of solids (Figure 2.12 (left)). A shear force F is applied on the upper plate with a shear area A while the lower plate is stationary (s = 0), resulting in a deflection s and a deflection angle φ. The sample is sheared in the shear gap z. If there are no wall-slip effects, the sample adheres to both plates, and if the sample is deformed homogeneously throughout the shear gap, the shear deformation or shear strain is defined as γ = 𝑠 𝑧⁄ = tanφ. (2.7) The shear rate γ̇ defined in Flow Behavior can be interpreted as the time derivative of γ, which writes γ̇ = 𝑑γ 𝑑𝑡⁄ [s-1]. (2.8) At a constant temperature, the ratio of the shear stress τ and the corresponding shear strain γ is a material constant if the measuring conditions are within the reversible-elastic deformation range (linear-elastic range). This ratio is called shear modulus or rigidity modulus G and gives information about the rigidity of a material. It holds 𝐺 = τ γ⁄ [Pa]. (2.9) If the molecules of the material have strong intermolecular or crystalline cohesive forces, it exhibits high rigidity and a high G-value. A z F s = 0 s φ Figure 2.12: Schematic presentations of (left) the Two-Plates-Model for elastic behavior, (right) the spring model. Theoretical Background 24 The ideally elastic (or Hookean) deformation behavior fulfills Hooke’s law, namely τ(𝑡) = 𝐺 ∙ γ(𝑡). (2.10) The deformation of an elastic solid is proportional to the applied force (shear stress) within the linear-elastic range, and the τ(γ)-function has a constant slope, i.e. the shear modulus G. The shear modulus is independent of the magnitude and duration of the applied shear load. The ideally elastic behavior can be illustrated by the spring model (Figure 2.12 (right)). If a constant force is applied, the spring deforms immediately and remains deformed as long as the force is present. The deformation disappears immediately after the removal of the force and returns to the initial state. In contrast to the dashpot model of the ideally viscous liquid, the spring has no viscous behavior at all. During the shear process, the applied deformation energy is entirely stored within the deformed material. The stored energy enables a complete reformation of the material once the load is removed. Thus, the deformation process is reversible for ideally elastic solids. The ideally elastic deformation behavior requires relatively strong interactions between atoms or molecules of the material, such as stone and steel. However, beyond the linear-elastic range, brittle fractures occur. Viscoelastic Behavior Viscoelastic materials exhibit both viscous and elastic behavior. The viscous portion follows Newton’s law, while the elastic portion behaves according to Hooke’s law. Viscoelastic materials show a time-dependent and delayed response to stress or strain when applied and removed. The rheological behavior of viscoelastic liquids differs from that of viscoelastic solids. To illustrate the difference, we use the Maxwell model to represent viscoelastic liquids and the Kelvin/Voigt model to represent viscoelastic solids. The Maxwell model combines a dashpot and a spring in serial connection (Figure 2.13 (left)). Upon applying a constant force, the spring deforms immediately and reaches a constant deflection value, which is proportional to the applied force. Subsequently, the piston of the Theoretical Background 25 dashpot starts to move with a constant velocity until the deformation reaches a constant value, which correlates to the applied force and the duration of time. When removing the force, the spring bounces back to its initial state immediately, while the dashpot remains at its deformed position. As a result, the elastic portion shows a reformation while the viscous portion leads to permanent deformation. The overall behavior resembles a liquid, thus the Maxwell model represents viscoelastic liquids. Figure 2.13: Schematic presentation of (left) the Maxwell model for viscoelastic liquids, (right) the Kelvin/Voigt model for viscoelastic solids. The Kelvin/Voigt model is a parallel combination of a spring and a dashpot connected by a rigid frame (Figure 2.13 (right)). Since the two components are connected by a rigid frame, they must move simultaneously and to the same extent. Therefore, upon applying a constant force, the spring cannot deform instantly. Instead, the shear strain increases as a time-dependent exponential function until it reaches the maximum. When removing the force, the spring tends to bounce back immediately. However, the dashpot slows down the process, leading to a time- dependent decreasing exponential function γ(t). After a sufficiently long time, the reformation completes, and both the spring and the dashpot go back to the starting position (γ = 0). Overall, the Kelvin/Voigt model displays a reversible but delayed deformation process, which behaves essentially like a solid. Thus, the Kelvin/Voigt represents viscoelastic solids. Oscillating Shear Rheometry Oscillatory shear rheometry is used to examine the rheological properties of all kinds of viscoelastic materials, including gels. Other types of rheometry, such as rotational rheometry that characterizes the flow behavior of liquids, are left out from this section since they are less Theoretical Background 26 relevant. The same shear conditions of the Two-Plates-Model as in Elastic Behavior are assumed: (1) There are no wall-slip effects, and the sample adheres to both plates. (2) The sample is deformed homogeneously throughout the shear gap. Additionally, the applied shear load (either shear stress or shear strain) oscillates sinusoidally, causing a deflection path of ±s, while the lower plate stays stationary (deflection s = 0, Figure 2.14). s = 0 s φ φ s + 0° 90° 180° 270° 90°270° Figure 2.14: Schematic presentation of the Two-Plates-Model for oscillatory tests. There are two preset modes for oscillatory tests: (1) Preset the shear strain γ as a sinusoidal function γ(𝑡) = γA ∙ sinω𝑡, (2.11) where γA is the shear strain amplitude, and ω is the angular frequency. This test method is called the controlled shear rate/deformation (CSR/CSD) test. (2) Preset the shear stress τ as a sinusoidal function τ(𝑡) = τA ∙ sinω𝑡 (2.12) with τA being the shear stress amplitude, which is called the controlled shear stress (CSS) test. Here we preset the shear strain γ as a sinusoidal function (CSR test) to deduce the response functions: (a) For ideally elastic behavior, Hooke’s law applies. Combining Equation 2.10 and 2.11, one obtains τ(𝑡) = 𝐺 ∙ γ(𝑡) = 𝐺 ∙ γA ∙ sinω𝑡. (2.13) Thus, there is no delay between the resulting γ(t)-curve and the preset τ(t)-curve, i.e., the phase shift angle (or loss angle) δ between the preset and the resulting curve is 0° (Figure 2.15). The Theoretical Background 27 shear rate is γ̇(𝑡) = 𝑑γ(𝑡) 𝑑𝑡⁄ = γA ∙ ω ∙ osω𝑡, (2.14) which is shifted by 90° compared to the γ(t)-curve. (b) For ideally viscous behavior, Newton’s law applies. Combining Equation 2.6 and 2.14, one obtains τ(𝑡) = η ∙ γ̇(𝑡) = η ∙ γA ∙ ω ∙ osω𝑡. (2.15) Therefore, both the γ̇(𝑡)-curve and the τ(t)-curve have a phase shift angle of δ = 90° compared to the preset γ(t)-curve (Figure 2.15). (c) For viscoelastic behavior, the τ(t)-curve has a phase shift angle δ compared to the preset γ(t)-function: τ(𝑡) = τA ∙ sin(ω𝑡 + ). (2.16) Overall, it always stands that for ideally elastic behavior δ = 0°, for ideally viscous behavior δ = 90°, and for viscoelastic behavior 0° < δ < 90° (Figure 2.15). Figure 2.15: The oscillating curves of ideally elastic, ideally viscous, and viscoelastic behaviors. Preset is a sinusoidal shear strain γ(t), and the shear stress τ(t) is the resulting response. δ ideally elastic ωt γ τ ideally viscous ωt γ τ viscoelastic ωt γ τ 90° Theoretical Background 28 Figure 2.16: The vector diagram of the complex shear modulus G*. Through the oscillatory tests, the complex shear modulus is obtained: 𝐺∗ = τ(𝑡) γ(𝑡)⁄ [Pa]. (2.17) On the complex plane in Figure 2.16, it gives 𝐺∗ = 𝐺′ + 𝑖𝐺′′, (2.18) with the imaginary unit 𝑖 = √−1. Then the real component 𝐺′ = 𝐺∗ ∙ os = (τA γA⁄ ) ∙ os (2.19) is the storage modulus, and the imaginary component 𝐺′′ = 𝐺∗ ∙ sin = (τA γA⁄ ) ∙ sin (2.20) is the loss modulus. The storage modulus G’ is a measure of the deformation energy stored by the sample during the shear process, which is the driving force for reformation when the load is removed. Thus, the storage modulus G’ represents the elastic behavior of the material. The loss modulus G’’ is a measure of the deformation energy consumed by the sample for frictional heating during the shear process and represents the viscous behavior of the tested material. The loss factor or damping factor is defined as tan = 𝐺′ 𝐺′′⁄ . (2.21) It reveals the ratio of the viscous and the elastic portion of the viscoelastic behavior. Since the Real Imaginary G* δ G’ G’’ Theoretical Background 29 phase shift angle has the range of 0° ≤ δ ≤ 90°, it holds 0 ≤ tan δ ≤ ∞. When a sol forms a gel, its elastic behavior exceeds the flow behavior. The sol–gel transition point is where the storage modulus G’ equals the loss modulus G’’, and it holds for the damping factor tan δ = 1 and the phase shift angle δ = 45°. For molecular gels, rheology is often used to characterize the linear viscoelasticity, the gel–sol transition temperature Tsol–gel, the kinetics of gelation, the material failure modes, and the timescale of viscoelastic recovery. In this thesis, rheological measurements are focused on the linear viscoelasticity and Tsol–gel. When the moduli are measured as a function of the applied shear strain or stress at a fixed frequency, gels typically show a linear regime or plateau called the linear viscoelastic range (LVE range) until the magnitude of the storage modulus G’ drops considerably and falls below the loss modulus G’’ (Figure 2.17). This measurement is called an amplitude sweep. In the LVE range, it always holds that G’ > G’’, and the power-law applies G’, G’’ ~ (γ, τ)0. Moreover, there is no significant internal structure change, i.e., the elasticity is reversible. The limiting point of the LVE range is called the yield point. At higher shear strains or stresses, the material loses its gel character. The point where G’ = G’’ is called the flow point, and the range between the yield point and the flow point is called the yield zone. In this range, although the sample still has gel behavior, the elasticity is irreversible. Nevertheless, instant recovery behavior has been observed for some molecular gels due to their physical bond reformation of the gel network [Ohs14, Mal16]. Theoretical Background 30 Figure 2.17: Schematic presentation of an amplitude sweep curve, showing a double- logarithmic scale of the storage modulus G’ and the loss modulus G’’ as a function of shear stress/strain. After having determined the LVE range from amplitude sweeps, frequency sweeps can be used to investigate the time-dependent deformation behavior of gels since the frequency is the inverse value of time. G’ and G’’ are measured at fixed shear stress or strain within the LVE range and constant temperature as a function of frequency. The short-term behavior can be obtained at high frequencies and the long-term behavior at low frequencies. For gels, it always holds G’ > G’’ over the entire frequency range, and the G’-curve and the G’’-curve are parallel to each other. In most cases, the power-law applies G’ ~ ωβ. Figure 2.18 (left) shows frequency sweeps of the organogel liquid paraffin/DBS at various gelator concentrations (G’’-curves omitted) [Liu13]. When the gelator concentration increases above 0.35 wt.%, the shape of the G’(ω)-curve is less steep compared to those at low gelator concentrations, and the magnitude of G’ is much larger, which means decently strong gels are formed. yield zone lgτ or lgγ lgG’ lgG” yield point flow point LVE range Theoretical Background 31 Figure 2.18: (left) Dependence of G’ on the angular frequency ω of the organogel liquid paraffin/DBS at various DBS concentrations [Liu13]. (right) Storage modulus G’ and loss modulus G’’ of the organogel n-decane/12-HOA with 1.5 wt.%, 2.5 wt.%, and 5.0 wt.% gelator as a function of temperature [Lau15]. The gel–sol transition temperature Tsol–gel can be determined by a temperature sweep. G’ and G’’ are measured as a function of temperature T at a fixed frequency and fixed shear stress or strain, which is within the LVE range. At low temperatures, it holds G’ > G’’ as the sample is a solid-like gel with elasticity. As the temperature increases, the gel starts to lose its elasticity and transforms into a less viscous liquid sol since gelator molecules are dissolved. At Tsol–gel, which is actually a temperature range of 5–10 °C, the storage modulus G’ drops sharply and crosses over with the loss modulus G’’. Increasing the temperature further, G’’ > G’, which means the viscous behavior dominates over the elastic behavior, and the sample becomes liquid. Figure 2.18 (right) shows temperature sweeps of the organogel n-decane/12-HOA [Lau15]. As described in Section 2.2, the Tsol–gel-value as well as the G’- and G’’-values increase with increasing gelator concentration. Methods 32 3 Methods 3.1 Chemicals, Sample Preparation and Visual Phase Studies Chemicals All chemicals used in this study are listed in Table 3.1. The chemicals were used without further purification. Plantacare contains a tiny amount of Mg2+ (< 500 ppm) and has basic pH values for preservative purposes, which caused the microemulsion samples to be slightly turbid. For a clear visual observation, the Plantacare surfactant was neutralized by adding citric acid anhydrous before use, leading to transparent microemulsions. Note that the effect of Mg2+ and citric acid on the phase behavior of microemulsions is negligible. Table 3.1: Chemicals used in this study. Name Abbreviation Supplier Purity water H2O bidistilled deuterium oxide D2O Eurisotpo > 99.9% sodium chloride NaCl Merck ≥ 99.5% n-octane Alfa Aesar > 98% 1-octanol Aldrich 99% isopropyl myristate IPM TCI > 98% 1,2-octanediol Acros > 98% n-octyl-β-D-glucopyranoside β-C8G1 GLYCON Biochemicals > 99.5% Plantacare 810 UP (technical- grade alkyl polyglucosides with an average composition of C8/10G1.5) BASF active content ~ 64%, water as solvent Plantacare 2000 UP (C10G1.5) BASF active content ~ 53%, water as solvent Plantacare 1200 UP (C12G1.4) BASF active content ~ 52%, water as solvent Methods 33 MEGA-8/10 (C8/10- methylglucamide) Clariant active content ~ 50% with 45% water and 5% propylene glycol MEGA-12/14-HC (C12/14- methylglucamide) Clariant active content ~ 64% with 22% water, 10% ethanol, and 4.5% propylene glycol MEGA-12/14-PC (C12/14- methylglucamide) Clariant active content ~ 35% with 60% water, 4% propylene glycol, and 0.8% sorbic acid citric acid anhydrous Sigma-Aldrich ≥ 99.5% 12-hydroxyoctadecanoic acid 12-HOA Alfa Aesar 95% N,N'-dibenzoyl-L-cystine DBC Santa Cruz > 98% 1,3:2,4-dibenzylidene-D- sorbitol DBS NJC Europe n/a 1,3:2,4-dibenzylidene-D- sorbitol–p,p’-dicarboxylic acid DBS–COOH synthesized by the group of Prof. Dr. David K. Smith from the University of York, UK n/a 1,3:2,4-dibenzylidene-D- sorbitol–p,p’-dihydrazide DBS– CONHNH2 lidocaine TCI > 99% diclofenac sodium salt Sigma-Aldrich ≥ 98% Sample Preparation Four kinds of samples were prepared: the non-gelled and gelled microemulsions for SANS measurements and phase studies. Non-gelled microemulsions for small-angle neutron scattering (SANS) measurements were prepared by weighing in surfactant (C), co-surfactant (D), oil (B), and water (A) in the mentioned order with an analytical balance into glass tubes, which were then sealed by polyethylene stoppers. Subsequently, the samples were heated up to 50 °C in a water bath while being stirred to homogenize. Afterward, the samples were equilibrated at 25 °C and then ready to use. For phase studies, the surfactant (C), oil (B), and water (A) were first weighed in, and the samples were homogenized at 50 °C. Afterward, phase studies were carried out at 25 °C titrating the co-surfactant (see Section Visual Phase Studies). Methods 34 The preparation of gelled microemulsions was similar to that of non-gelled microemulsions, but a gelator was additionally added to the samples. The samples were firstly heated above 92 °C to dissolve the gelator. A heat gun was utilized when necessary. Subsequently, the samples were quickly transferred to an ice bath directly from the 92 °C water bath with manual shaking to ensure homogeneous gelation. Afterward, the samples were transferred to a 25 °C water bath and allowed to equilibrate. Sample compositions were defined by the volume fraction of oil (B) in the solvent mixture ϕ = 𝑉 𝑉w + 𝑉 (3.1) the mass fraction of surfactant (C) γC = 𝑚 f. 𝑚 (3.2) and the mass fraction of the co-surfactant (D) γD = 𝑚c f. 𝑚 . (3.3) When using technical-grade surfactants, 𝑚 f. is the weight of the active component in the original surfactant formulation, and 𝑚 is the total mass of water, oil, the total mass of the technical-grade surfactant (including solvent and additives), co-surfactant, and gelator (if the sample is gelled microemulsion). When salt was added, the salinity of the microemulsion sample is defined as ε = 𝑚 𝑚w +𝑚 . (3.4) In the case of gelled microemulsions, the gelator concentration is calculated according to η = 𝑚 𝑚 . (3.5) Methods 35 Visual Phase Studies The microemulsion phase behavior was investigated at a constant temperature of T = 25.0 °C using a house-built setup (Figure 3.1). The water basin was equipped with a thermostat and a cooling system to maintain a constant temperature with a precision of ± 0.1 K. The non-gelled microemulsion samples were firstly prepared without co-surfactant (D). After equilibration in the water bath, the stopper was removed, and a co-surfactant (D) was titrated to the sample dropwise by a syringe. The added amount of co-surfactant was recorded using an analytical balance with a precision of ± 0.001 g. After adding the co-surfactant, the sample was shaken and stirred vigorously before it was left to equilibrate. Then the phase behavior was visually determined. Birefringence of lyotropic liquid crystalline phases was observed via crossed polarizers. Since the phase transitions of sugar surfactant-based microemulsions were induced by the addition of a co-surfactant, the phase behavior was presented in a phase tetrahedron (Figure 2.3). The phase behavior of these microemulsions was studied at a constant oil-to-water ratio, i.e., recording a two-dimensional section through the tetrahedron. After measuring the phase boundaries, 1-phase microemulsion samples of different oil-to-water ratios (ϕ) were prepared to study the gelation behavior of LMWGs. We first determined the minimum gelation concentration (mgc) by adding a gelator to the microemulsion sample until the whole sample was gelled. The sample is considered gelled if the material does not flow in the inverted test tube. After knowing the mgc, the phase behavior of gelled microemulsions can be measured. The gelled microemulsion sample was first prepared without a co-surfactant, then the co-surfactant was titrated dropwise to the gelled sample at 25 °C. The sample was then heated above the gel–sol transition temperature, where the sample was melted and could thus be mixed. After the gelation process described in Section Sample Preparation, the phase behavior was recorded. A gelled 1-phase microemulsion is transparent, while a gelled 2-phase microemulsion is turbid. The titration, melting, and gelation procedures were repeated until the gelled microemulsion sample crossed the 2–1 and 1–2 phase boundaries. Methods 36 Figure 3.1: The house-built experimental setup for visual phase studies: A – thermostat, B – water basin, C – microscopy lamp, D – crossed polarizers, E – digital thermometer, F – magnetic stirrer, G – house-made sample holder with the sample, H – co-surfactant bottle with a syringe, I – balance for recording the amount of added co-surfactant. 3.2 Oscillatory Shear Rheometry Oscillatory shear rheometry was carried out to characterize the rheological properties of gelled microemulsions. The characteristic rheological behavior of molecular gels is described in Section 2.3. We used the rheometer Physica MCR 501 from Anton Paar, Austria, and a plate– plate geometry with the upper plate of 25 mm diameter. The gap size between the two plates was chosen to be 1 mm according to a previous study [Lau13]. The temperature was controlled via an external thermostat with a precision of ΔT = ± 0.1 K. Firstly, an amplitude sweep was carried out at T = 25 °C with an angular frequency of ω = 10 s-1 and a controlled shear stress τ to determine the limit of the linear viscoelastic range (LVE range). The shear stress for the subsequent measurements should be within the limit of the LVE range. Hence, for the gelled Methods 37 microemulsions, the shear stress was kept constant at τ = 10 Pa except for the samples with ϕ = 0.20 and ϕ = 0.35 at η = 0.002 in Publication IV, which were measured at a shear stress of τ = 1 Pa. Secondly, an oscillation frequency sweep was performed at T = 25 °C as a function of the angular frequency ω. Lastly, a temperature sweep was carried out at an angular frequency of ω = 10 s-1 from T = 25 °C to T = 100 °C with a heating rate of 1 K∙min-1 to determine the gel–sol transition temperature Tsol–gel. Note that the storage modulus G’ and the loss modulus G’’ were measured only once for each sweep due to large deviations (10 – 50%) of repeated measurements [Ste19, Ste19b]. Thus, the absolute values should be considered carefully while the trends are discussable. We distinguish between “strong” and “weak” gel by the magnitude difference of the storage modulus G’ and the loss modulus G’’. A sample is considered to be “rigid” if it is frequency-independent and “soft” if frequency-dependent. 3.3 Small-Angle Neutron Scattering Small-angle neutron scattering (SANS) provides quantitative characterizations of the microstructure and the length scale of microemulsions [Hel08]. For the sample preparation, we replaced H2O with D2O to adjust bulk contrast. The phase boundaries of the deuterated system were measured again using the titration procedure, and the SANS sample was prepared in a composition close to the middle of the 1-phase region. Both non-gelled and gelled microemulsions were first prepared in test tubes and then loaded into Hellma quartz QS glass cells (optical path length of 1 mm). The non-gelled microemulsions were transferred to the measuring cell at T = 25 °C in the 1-phase state. The gelled microemulsion was transferred at T = 95 °C with stirring so that the sample was in the sol state and a homogeneous mixture, and the cell was rapidly cooled down in an ice bath and then equilibrated at T = 25 °C to form a transparent gel. Before the SANS measurement, the measuring cell was transferred to a house- built cell holder with high temperature precision and stability (ΔT = ±0.02 K). It was ensured that samples were in the homogeneous 1-phase state during measurements by checking each sample before and after the measurement via visual inspection. Methods 38 Figure 3.2: Schematic illustration of the instrument layout for SANS (taken from [www01]). In Publication I, the glucose-containing non-gelled and gelled microemulsions were measured on the D11 spectrometer at the Institut Laue-Langevin (ILL) in Grenoble, France. The instrument layout is depicted in Figure 3.2. A neutron wavelength of λ = 5.5 Å with a wavelength spread of Δλ/λ = 9% (full width at half-maximum) was used. To cover a q-range from 0.0016 to 0.47 Å, where 𝑞 = 4𝜋 ∙ sin(𝜃 2⁄ )/𝜆 (3.6) is the absolute value of the scattering vector, we chose the detector/collimation distances of 39.0 m/40.5 m, 8.0 m/8.0 m, and 1.5 m/20.0 m. The recorded scattering intensity was normalized to absolute scale using the incoherent scattering of H2O as a reference with a differential scattering cross section of 0.956 cm-1 at λ = 5.5 Å. The raw data treatment, including the subtraction of the dark current and the empty cell scattering, masking, and radial averaging, was performed using the analysis software program LAMP provided by the ILL. The detector dead time and sample transmission were also considered. The measurements were performed by our colleagues, Kristina Schneider, Shih-Yu Tseng, and Diana Zauser. In Publication II, the glucamide-containing microemulsions were measured on the spectrometer NG7 at the National Institute of Standards and Technology (NIST), the USA, and on the instrument KWS-1 at the Heinz Maier-Leibniz Zentrum (MLZ) in Munich, Germany. The microemulsion samples of γC = 0.0521, γD = 0.0878 with ε = 0 and γC = 0.0689, γD = 0.0927 with ε = 0.0009 were measured on NG7, NIST. A neutron wavelength of λ = 6 Å with Methods 39 a wavelength spread of Δλ/λ = 13.8% was used. We chose the detector/collimation distances of 13.17 m/14.72 m, 4 m/8.52 m, and 1.33 m/5.42 m to cover a q-range from 0.002 to 0.48 Å. The microemulsion samples of γC = 0.0232, γD = 0.0737 with ε = 0 and γC = 0.0522, γD = 0.0878 with ε = 0.0009 were measured on KWS-1, MLZ. Neutron wavelengths of λ = 5 and 10 Å with a wavelength spread of Δλ/λ = 10% were used to cover a q-range from 0.001 to 0.66 Å. The detector/collimation distances were 20 m/20 m, 8 m/20m, and 1.5 m/8m. The recorded scattering intensity was normalized to the absolute scale using the empty beam (for the data from NIST) and the plexiglass (for the data from MLZ) as a reference. The raw data treatment, including the subtraction of the dark current and the empty cell scattering, masking, and radial averaging, was performed using IGOR Pro (for the data from NIST) and QtiSAS (for the data from MLZ). The detector dead time and sample transmission were also considered. The measurements were performed by our colleagues, Shih-Yu Tseng, Karina Abitaev, and Diana Zauser. Obtained data were fitted with the Teubner–Strey model [Teu87], which describes the characteristic peak of a bicontinuous microemulsion with 𝐼(𝑞) = 8𝜋𝑐2𝜙 𝜙b(∆𝜌) 2/𝜉TS 𝑎2 + 𝑐1𝑞2 + 𝑐2𝑞4 + 𝐼 nc h (3.7) where Iincoh is the incoherent background, ∆𝜌 is the scattering length density difference of the two subphases, and 𝜙 and 𝜙b are their respective volume fractions. The parameters 𝑎2, 𝑐1 and 𝑐2 are coefficients derived from a Landau–Ginzburg order parameter expansion of the local free energy density, where the order parameter is the water-to-oil ratio. After determining the values of 𝑎2, 𝑐1 and 𝑐2, two characteristic length scales of the Teubner–Strey model can be calculated, namely the correlation length 𝜉TS = [ 1 2 ( 𝑎2 𝑐2 ) 1 2 + 𝑐1 4𝑐2 ] 1 2 (3.8) and the periodicity of the oil and water domains, i.e. the domain spacing Methods 40 𝑑TS = 2𝜋 [ 1 2 ( 𝑎2 𝑐2 ) 1 2 − 𝑐1 4𝑐2 ] 1 2 . (3.9) The amphiphilicity factor [Sch94] can be calculated by 𝑓 = 𝑐1 √4𝑎2𝑐2 . (3.10) 3.4 Freeze-Fracture Electron Microscopy The liquid mixtures of a microemulsion can be solidified via cryofixation, and then the microstructure can be visualized by transmission electron microscopy (TEM). In freeze- fracture electron microscopy (FFEM), the samples are prepared in a protected fashion in a sandwich. Subsequently, they are rapidly frozen, fractured, shadowed with metal, and replicated with a thin carbon film. The replica of the fractured surface is then studied by TEM [Sot08]. The sample preparation and operation of FFEM are sophisticated and demand rich experience to obtain decent images. Thus, the FFEM images were taken by Dr. Natalie Preisig, an expert in this field. The experimental details are briefly described here. Replicas of gelled and non-gelled microemulsions were prepared using the Freeze-Fracture and Etching System BAF060 from Leica. A small amount of the gelled microemulsion was transferred from the test tube to two copper grids between two copper plates (4.5 mm × 3.0 mm), which were then assembled into a so-called sandwich. In the case of non-gelled microemulsion, the sandwich was immersed in the liquid sample with a tweezer for several minutes. Afterward, the sandwiches were quickly frozen in liquid ethane. After fracturing in liquid nitrogen, the grids with the frozen fractured specimen were fixed on a house-made specimen holder (with a metal cover plate to prevent contamination in the air) and quickly transferred into the vacuum chamber of the BAF060 (T = –150 oC). The frozen fractured surface of the specimens was shadowed with platinum-carbon (~2 nm) at 45° and covered by a layer of pure carbon (~20 nm) at 90°. The replicas were then cleaned with warm ethanol and Methods 41 acetone, dried, and examined with an EM10 transmission electron microscope from Zeiss operated at 60 kV. 3.5 Electrical Conductivity Electrical conductivity measurements were performed to monitor the structural change from water-rich to oil-rich microemulsions in Publication IV. Since water domains with salt have high electrical conductivity while oil domains have low conductivity, the structural change can be observed by the conductivity change [Sot05]. The experimental setup consisted of the conductivity cell TetraCon 925/LV from WTW (Xylem Analytics GmbH, Weilheim, Germany) integrated with a temperature sensor and the conductivity meter Multi 3510 IDS from WTW. The conductivity measuring cell was placed in a test tube, which contained the sample to be measured. The temperature was adjusted to a constant value of T = (25.0 ± 0.1) °C by placing the test tube in a water bath regulated by a thermostat DC30 from Thermo Electron GmbH (Karlsruhe, Germany). For a detectable conductivity, the water of the microemulsion sample was replaced by 0.1 wt.% of NaCl solution. Note that the effect of salt on the phase behavior of nonionic microemulsions is negligible at this low concentration. The electrical conductivity was measured through the titration procedure as described in Section 3.1. The sample was continuously stirred with a Teflon-coated magnetic stirring bar, and the co-surfactant was added to the sample dropwise. Thus, κ(γD)-curves were established by recording the conductivities after each drop of co-surfactant and 3 min of waiting time. Summary of Research 42 4 Summary of Research The results have been published in four publications [Pen19 (Publication I), Pen20 (Publication II), Pen21a (Publication III), Pen21b (Publication IV)]. In Publication I & II, we formulated non-toxic bicontinuous microemulsions using different types of surfactants, including alkyl polyglucosides (APGs) and alkanoyl methylglucamide (MEGA), and identified a suitable low molecular weight gelator (LMWG) for gelling non-toxic microemulsions. In Publication III, we loaded both hydrophilic and hydrophobic model drugs in the gelled non-toxic microemulsion for its potential application as a transdermal drug delivery carrier. In Publication IV, we studied the non-toxic microemulsions at various oil-to-water ratios and identified suitable LMWGs for both water-rich and oil-rich microemulsions. These studies broaden the application potentials of gelled non-toxic microemulsions and reveal information about the interactions between gelators and solvents (in our case, microemulsions). The most important findings and the connections between these publications will be described in the following sections. 4.1 Gelled Non-Toxic Microemulsions: Phase Behavior & Rheology (Publication I) We intended to formulate non-toxic microemulsions with biocompatible sugar surfactants, namely alkyl polyglucosides (APGs, denoted as β-CnGm, see Figure 1.3 for molecular structure) [Stu01]. The phase behavior, interfacial composition, interfacial tension, and microstructure of the quaternary microemulsion H2O – n-octane – n-octyl-β-D-glucopyranoside (β-C8G1) – 1-octanol were extensively studied before [Klu00, Klu01, Sot02, Rei03]. Thus, we used this system as the scouting system and replaced both the oil n-octane and the co-surfactant 1-octanol with non-toxic components. Our focus was on the location of the fishtail point �̃� (where the 1- phase region meets the 3-phase region), where the microstructure is bicontinuous. Summary of Research 43 Figure 4.1: (left) Phase diagrams of the quaternary systems H2O – n-octane – β-C8G1 – 1- octanol (gray circles) and H2O – n-octane – β-C8G1 – 1,2-octanediol (black triangles up) at T = 25 °C, ϕ = 0.50. (right) Phase diagrams of quaternary system H2O – n-octane – β-C8G1 – 1,2- octanediol (black triangles up), and H2O – IPM – β-C8G1 – 1,2-octanediol (green diamonds) at T = 25 °C, ϕ = 0.50. Reproduced from [Pen19] with permission from the Royal Society of Chemistry. We restudied the phase behavior of the scouting system H2O – n-octane – β-C8G1 – 1-octanol (Figure 4.1 (left), grey circles). One sees that the addition of 1-octanol induces phase transitions in the sequence of 2 – 3 – 2 and 2 – 1 – 2, respectively. Then we replaced the co-surfactant 1-octanol with the biocompatible 1,2-octanediol (Figure 4.1 (left), black triangles up). 1,2- Octanediol is much more hydrophilic than 1-octanol, and its effect on the phase behavior is similar to that of 1-butanol [Kah96]. Thus, the amount of 1,2-octanediol needed to form a 1- phase microemulsion γ̃C is considerably higher than that of 1-octanol. However, since the oil component remains the same, the amount of β-C8G1 needed to form a 1-phase microemulsion γ̃D does not change (�̃� points of the two systems are shifted parallel to the co-surfactant axis). The more hydrophobic 1,2-dodecanediol was also examined. However, the sample solidified at 25 °C due to its high melting point. Subsequently, n-octane was replaced by isopropyl myristate (IPM, Figure 4.1 (right)). The shift of the �̃� point to the right upper side of the phase diagram can be explained by two reasons: (1) IPM has a longer carbon chain compared to n-octane, which decreases the efficiency of the surfactant (an increase in γ̃D). (2) 1,2-Octanediol has a higher solubility in IPM than in n-octane, so a slightly higher amount of 1,2-octanediol is H 2 O/n-octane f = 0.5, T = 25.0 °C 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.05 0.10 0.15 b-C 8 G 1 co-surfactant 2 _ 2 0.20 0.25 1 3 L a H 2 O/oil f = 0.5, T = 25.0 °C 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.05 0.10 0.15 b-C 8 G 1 1,2-octanediol 2 _ 2 0.20 0.25 1 3 Summary of Research 44 needed (an increase in γ̃C). Figure 4.2: (left) Phase diagrams of H2O – IPM – β-C8G1 – 1,2-octanediol (green diamonds) and H2O – IPM – Plantacare 810 UP – 1,2-octanediol (dark green squares) at T = 25 °C, ϕ = 0.50. (right) Phase diagrams of H2O – IPM – Plantacare 810 UP (C8/10G1.5) – 1,2-octanediol (dark green squares), H2O – IPM – Plantacare 2000 UP (C10G1.5) – 1,2-octanediol (cyan triangles down), and H2O – IPM – Plantacare 1200 UP (C12G1.4) – 1,2-octanediol (blue hexagons) at T = 25 °C, ϕ = 0.50. Reproduced from [Pen19] with permission from the Royal Society of Chemistry. Since the efficiency (γ̃C + γ̃D) of the non-toxic microemulsion H2O – IPM – β-C8G1 – 1,2- octanediol largely decreased compared to the scouting system H2O – n-octane – β-C8G1 – 1- octanol and because the pure surfactant β-C8G1 is quite expensive, the next step was to replace β-C8G1 with technical-grade APGs. Firstly, β-C8G1 was replaced by Plantacare 810 UP (C8/10G1.5, Figure 4.2 (left)). One sees that the �̃� point is slightly shifted to a position where less surfactant and co-surfactant are needed. It also indicates that the longer carbon chain of Plantacare 810 UP compensates for its larger number of glucoside units, and the overall efficiency is slightly improved. In order to further increase the efficiency, technical-grade APGs with longer carbon chains were investigated (Figure 4.2 (right)), including Plantacare 2000 UP (C10G1.5) and Plantacare 1200 UP (C12G1.4). One sees that the longer the carbon chain is, the more efficient the system becomes. Simultaneously, the required co-surfactant amount decreases considerably due to the increased surfactant hydrophobicity. Therefore, the non-toxic H 2 O/IPM f = 0.5, T = 25.0 °C 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 b-C 8 G 1 Plantacare 810 UP 0.05 0.10 0.15 sugar surfactant 1,2-octanediol 2 _ 2 0.20 0.25 1 3 H 2 O/IPM f = 0.5, T = 25.0 °C 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 810 UP 2000 UP 1200 UP 0.05 0.10 0.15 Plantacare