Self-Assembly of Amphiphiles into Vesicles and Fibrils - American

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Self Assembly of Amphiphiles into Vesicles and Fibrils: Investigation of Structure and Dynamics using Spectroscopic and Microscopic Techniques Niloy Kundu, Debasis Banik, and Nilmoni Sarkar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04355 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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Self-Assembly of Amphiphiles into Vesicles and Fibrils: Investigation of Structure and Dynamics using Spectroscopic and Microscopic Techniques Niloy Kundu, Debasis Banik and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303 Abstract Amphiphiles are class of molecules which are known to assemble into a variety of nanostructures. The understanding and applications of self-assembled systems are based on what has been learned from biology. Among the vast number of self-assemblies, in this article, we have described the formation, characterization and dynamics of two important biologically inspired assemblies, vesicles and fibrils. Vesicles, which can be classified into several categories depending on the sizes and components, are of great interest due to their potential application in drug delivery and as nanoscale reactor. The structure and dynamics of vesicles can also mimic the complex geometry of cell membrane. On the other hand, self-assembly of proteins, peptides and even single amino acids lead to number of degenerative disorders. Thus, the complete understanding of these self-assembled systems is necessary. In this article, we discuss the recent work on the vesicular aggregates composed of phospholipids, fatty acids and ionic as well as non-ionic surfactants and single amino acid based fibrils, such as phenylalanine, tyrosine etc. Beside the characterization, we also emphasize on the excited state dynamics inside the aggregates for the proper understanding of the organization, reactivity, and heterogeneity of the aggregates.

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1. Introduction. All living organisms in earth are composed of biological cells and this is the most generalization cell theory of life in biology.1The foundation of modern research on origin of life is based on the idea of molecular evolution as proposed by Oparin, several years ago.2 According to Oparin, life originated from non-living molecules through spontaneous increase of molecular complexity and specificity. This hypothesis considers “amphiphilicity” as one of the critical property for the development of “compartmentalization".2,3 It is a semi-permeable membrane which controls the flow of the materials in and out of the cell and acts as a selective barrier between the interior of the cell and outer external environment. Modern cell membranes are composed of mixture of different amphiphiles such as phospholipids, glycolipids, fatty acid (FA) chain which contain hydrophilic head group and hydrophobic acyl chains.4The hydrophobic bilayer which is few nanometer thin form a strong barrier to ion and different polar molecules and maintains nonequilibrium concentration of the molecules in the interior of the cell by preventing the free diffusion between the inner and outer compartment. The transfer of ions through the bilayer also involves different membrane proteins which assist in the movement of the substances through facilitated diffusion or active transport.3Moreover, the cellular growth and division depend on several critical biochemical reactions which possibly arose during the cellular evaluation. However, early cell membranes are thought to comprise of simple single chain amphiphiles, such as fatty acids. As a consequence, a lot of studies have been devoted to understand the growth and division of cell membrane by considering the simple FA based vesicles or simple amphiphilic vesicles as a model membrane to avoid the advanced biochemical machinery.5,6The microstructure of the vesicles can mimic the complex structure of the cell membrane in which the plasma membrane has been replaced by the abiotically formed simple amphiphilic molecules. Phosphoplipids or other amphiphiles spontaneously form spherical vesicles in water which are delineated by bilayer membrane. The diameter of the bilayer is approximately few nanometers depending on their geometry. The bilayer structure of vesicles composed of oleic acid (OA) and linoleic acid was first recognized by Gebicki and Hicks by electron microscopy using freeze etching technique7 and it leads to the beginning of the detailed studies on the structural properties of biomembranes and mimicking their functions using a variety of synthetic amphiphiles. The 2 ACS Paragon Plus Environment

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aggregates are formed due to the balance between the electrostatic attractive and repulsive forces. Moreover, the hydrophobic entropic effect is also reasonable factor for the formation of such assemblies. The aggregation of amphiphile’s tail groups disrupt the strong hydrogen bonding interactions between water molecules and lower the free energy. Besides, the coulombic repulsion between the head groups of the amphiphiles interdicts the phase separation of the aggregates. Among the amphiphiles, phospholipids are most abundant in nature. However, these vesicles are highly unstable and their applicability is limited by the hydrolysis and oxidative degradation of phospholipids.8 Thus, the search for alternative vesicle with enhanced stability is a long standing topic of current research and the last few years have witnessed the revolutionary movement of drug delivery in biomedical research by developing different non-phospholipid based vesicles which are composed of different common surfactants and amphiphiles.9,10Recent studies suggest that vesicle composed of biologically acceptable non-ionic surfactant and polymers which are known as noisome or polymersome are very effective for the drug delivery.11,12 Beside the surfactant’s self-assemblies, nature also endows life with a variety of synergistic and sophisticated protein aggregation (amyloids). Although vesicles are served as a carrier of different molecules into the cells, amyloids are associated with various disorders (especially neurodegenerative) such as Alzheimer’s disease, transmissible spongiform encephalopathy, type II diabetes, and prion disorders.13-15 Until the first decade of 21st century (up to 2012), the amyloid formation was restricted to proteins and peptides. In 2012, Gazit and coworkers have shown for the first time that Phenylalanine (Phe), as a single amino acid, forms fibrils which have similar morphology, cytotoxicity, electron diffraction pattern and dye-binding property like other amyloids.16This was one of the most important discoveries in amyloid research as the exact reason of aggregation has been identified after extensive investigation. Recently, Shaham-Niv et al. have found that several metabolites such as adenine, orotic acid, cystine, uracil, uric acid etc. form similar amyloid like aggregates.17Among these, self-assembled structures of L-Phe and LTyr are mostly focused. L-Phe is one of the essential amino acids in human. The first step of LPhe metabolism involves the conversion of L-Phe to L-Tyr through an enzyme phenylalanine hydroxylase (PAH). The next step is the degradation of L-Tyr into various small molecules through five enzymatic steps; among them the last step involves an enzyme tyrosine transaminase (TT). Genetic mutation of these two enzymes (PAH and TT) increases the 3 ACS Paragon Plus Environment

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concentration of L-Phe and L-Tyr in blood from normal to toxic level. Excess L-Phe and L-Tyr deposit in different portions of brain tissue, plasma and cerebrospinal fluid and thus interrupt proper brain development. As a consequence, two metabolic disorders are generated called Phenylketonuria (PKU) and Tyrosinemia type II.17,18 Literature reports have suggested that in case of hyperphenylalaninemia and tyrosinemia type II patients the blood Phe and plasma Tyr concentrations are 1.2 and 1.4 mM, respectively.18 It is to be noted that these concentrations are 20 times greater than the normal ranges and using lipid model membrane it is showed that Phe can increase the membrane permeability, which is mainly responsible for some of the harmful symptoms associated with high biological phenylalanine concentrations which occur with the genetic disorder PKU.19 Molecular dynamics simulation and infrared spectroscopic results indicate that hydrogen bonding and electrostatic forces are the major contributing factors in the formation of L-Phe fibrils.20 In a recent study, Mossou et al. solved the crystal structure of L-Phe self-assembly at neutral pH.21 They have reported that the self-assembly of L-Phe is guided by the π-π stacking and hydrogen bonding interactions between −NH3+ and –COO- groups. Every molecule of L-Phe participates in four intermolecular hydrogen bonding within the crystal. This feature article outlines the recent achievements and new trends of self-assemblies of different amphiphiles forming vesicles and fibrils.

Some reliable microscopic, as well as

spectroscopic techniques for the characterization of these self-assembled systems are discussed. Besides, different dynamic phenomena inside the amphiphilic aggregates have been discussed using different hydrophobic as well as hydrophilic dye molecules. Depending on their hydrophobicity they are located in different regions of the aggregates and this behavior is very similar to the biological assemblies where different bioactive molecules are distributed in different regions of the complex cellular environments. These complex behaviors inside the aggregates can be monitored by measuring the water dynamics, fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) techniques. Thus, given all the dominant advantages hinted by natural self-assembly, formation of different self-aggregated structures, such as vesicle, fibril provides better understanding of biological assemblies. In turn, the investigation of the complex structural details and kinetics/ thermodynamics control of theses assemblies provide deep insight into the formation of aggregates.


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2. Classification of Vesicles. In general, vesicle can be classified either based on their structural parameters (such as size, number and bilayer property etc.) or on the basis of their preparation methods. Depending on the number of bilayers they can be classified as unilamellar, oligolamellar, multilamellar etc. Oligolamellar vesicles (OLVs) and multilamellar vesicles (MLVs) have membrane making up of several and multiple bilayers respectively and the bilayers are separated by water molecules (table 1).These classifications are stated in detail in literature.32 However, vesicle can also be categorized based on their components i.e. the nature of amphiphiles. The structure of the amphiphilic molecules determines the stability as well as dynamics of the vesicles. In the next section, we will describe the detailed characterization, utility and dynamics of vesicles prepared from different amphiphilic molecules. Table 1. Classification of vesicles based on their size and components.

Based on Components

Vesicles

Components

Phospholipid Vesicles

Phospholipids

Fatty Acid (FA) Vesicles

Fatty acids (oleic acid, linoleic acid etc.)

Niosomes

Non-ionic surfactants (pluronic, tween, brij, span derivatives) and cholesterol or PEG mixture Mixture of differently charged surfactants or mixture of surfactants and Amphiphiles

Micelle to Vesicle transition by different amphiphiles

Based on Structural Parameters

MLV OLV UV SUV LUV GUV

Multilamellar large vesicle, > 0.5 Oligolamellar vesicle, 0.1-1 Unilamellar vesicle, all sizes Small unilamellar vesicle, 20-100 Large unilamellar vesicle, >100 nm Giant unilamellar vesicle, > 1


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2.1. Phospholipid Vesicles. The major components of modern cell are double chain amphiphiles, specifically phospholipids. Phospholipids are double chained amphiphiles containing negatively charged phosphate ester head groups which are coupled to different polar functional groups, such as choline, serine, glycerol, ethanolamine etc. Due to their geometry they spontaneously form vesicle in water. However, due to their slow dynamics they pose several fundamental problems as a protocell. The permeability of these vesicles to different charged molecules is very slow and the rate of flip-flop of these vesicles are also slow compared to FA vesicle.23Depending on the alcohol group phospholipids can be classified as glycerophospholipids and sphingogomyelins (SMs). Variation in the head group structure leads to the formation of different glycerophospholipids, such as phosphatidylcholine

(PC),

phosphatidylethanolamine

(PE),

phosphatidylserine

(PS),

phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylglycerol (PG) etc. The chain length of apolar moieties characterizes different glycerophospholipids, such as, dipalmitoyl, dimyristoyl, distearoyl PC etc. These vesicles are extensively used for drug delivery or gene delivery as they have the unique ability to solubilize both hydrophilic and hydrophobic molecules.24In recent times, much effort has been made to understand the structure and dynamics of phospholipid-based vesicles and it is discussed in later sections. 2.2. Fatty Acid Vesicle. Fatty acid vesicles are considered as one of the important biomimetic model membrane systems due to their chemical simplicity.25The most important feature of the FA vesicle is the selfreplication.25,26Thus, it has great relevance to understand the emergence of cellular life in real biological system. Therefore, the self-assembly of FA vesicle is a great paradigm of origin of life and protocell membrane.27,28Compared to phospholipid vesicles, FA based vesicles are better suited as a model membrane as the bilayer permits diffusion of the different ions and small molecules in and out of the vesicle compartment. FAs are single chain amphiphile and the concentration of the non-associated monomers in equilibrium with FA vesicles is much higher compared to that in phospholipid vesicles. For this reason, the rate of flip-flop in FA vesicles is expected to be much faster.23Extensive studies have been performed to understand the small molecule diffusion, competitive growth and division of vesicles and membrane mediated pH


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gradient etc.29,30 Further, the giant fatty acid vesicles are used to incorporate the drug, proteins and various biological macromolecules and it has gained significant interest in recent years.31,32 Fatty acids such as, OA, linoleic acid form different supramolecular structure depending on the pH of the medium. (figure 1(a)).33For example, the apparent pKa value of OA is 8. Thus, when the pH of the medium is maintained greater than 8, sodium oleate is the dominant component. However, at pH6, cholesterol must be added for the formation of vesicles. However, for surfactants with lower HLB, cholesterol enhances the stability of vesicle by promoting the gel-liquid transition temperature of the vesicle. Cholesterol increases the chain order of liquid state bilayer. However, it decreases the chain order of gel state bilayer and the gel state is transformed into liquid-ordered phase at high cholesterol content. With increasing concentration of cholesterol in the bilayer the release rate of encapsulated material is decreased. Therefore, the rigidity of the bilayer is increased. It increases the interlamellar distance between the successive bilayers of vesicles which leads to greater overall entrapped volume. Roy et al. have investigated the formation of pluronic triblock copolymer (F127)-cholesterol based niosomes and their interaction with different sugar molecules (figure 2).53The formation of niosomes was systemically characterized by dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) measurements. F127 forms spherical micellar aggregates in water. The transition of F127 micellar aggregates into noisomes with addition of cholesterol was monitored by DLS measurement. The size of the F127 micelle is ~27 nm and addition of cholesterol enhances the size of the aggregate upto 100-500 nm. They have further investigated the effect of sucrose on the niosome structure. In lipid bilayer, the presence of sugar molecules causes the head group to be separated. Hence, the lateral area is increased.54 In case of niosome, they have also obtained similar result. With addition of sugar, the size of the aggregates was increased which was reflected in both DLS and TEM measurements. Mandal et al. have also successfully formulated niosome composed of tween-20 and cholesterol.49 Similar to F127, tween-20 also forms spherical micelle in aqueous solution of 7 nm size. With addition of cholesterol, the micellar aggregate is turned into niosome of size 80-200 nm as evidenced by TEM measurements. Diblock copolymers modified with polypeptide can also form vesicle type structures in water.55 PEG can also be employed for the preparation of niosomes. Ghatak et al. have formulated niosomes composed of tween-80 and PEG 6000.50Tween-80 and PEG 6000 form lamellar liquid crystal in water after proper mixing and niosome is obtained after sonication of the diluted liquid crystal solution for thirty minutes. The presence of PEG on the niosome surface provides the strong interbilayer repulsion and it can overcome the attractive van der waals forces. Therefore, PEG stabilizes niosome by avoiding aggregation. Thus, the permeability and stability properties of niosomes can be altered by manipulating membrane characteristics by different additives. 11 ACS Paragon Plus Environment

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Figure 2. Schematic representation of formation of noisome composed of triblock copolymer (F127)/ cholesterol and effect of sucrose on noisome. The locations of donor and acceptor molecules for FRET are shown. Reproduced with permission from ref. 53. (Copyright 2016, American Chemical Society). Niosomes have attracted a great attention in controlled drug delivery system because of their biocompatibility, non-imminogenicity nature. It can encapsulate various drug molecules, which includes doxorubicin, insulin, ovalbumin, EGFP, DNA vaccine,

interferon and many others.

Further, niosomes have been applied to various delivery routes such as, intravenous, intramuscular, oral, ocular, pulmonary, transdermal etc.52Thus, niosomes present a convenient, prolonged, targeted and effective drug delivery system with the ability to load both hydrophilic and lipophilic drugs. 2.4. Micelle to Vesicle Transition by different Amphiphiles. In recent times, mixture of two oppositely charged surfactants, cationic surfactants have attracted a lot of attention because of their pharmaceutical applications, nano particle synthesis. The complex which is composed of two oppositely charged amphiphiles is referred as ion pair amphiphile (IPA). Both of the cationic surfactant and IPA can form vesicular aggregates in water.9, 56-59In general, a long chain surfactant can form vesicle in aqueous solution by mixing with oppositely charged surfactants or organic additives. The transition from micelle to vesicle in aqueous solution is a unique phenomenon which mimics different biological processes.60The 12 ACS Paragon Plus Environment

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morphology of aggregates of two oppositely charged surfactants is depended on several factors, such as the geometry of the surfactant molecule, pH of the medium, temperature, and ratio of mixing of surfactant molecules.61-63 Therefore, in the mixture of two differently charged surfactant molecules, a broad range of aggregates like micelles, mixed micelles (disk, ellipsoidal, sphere or wormlike) and vesicles (unilamellar as well as multilamellar) are found in aqueous solution. Kaler et al. first reported the formation of spontaneous, single walls, equilibrium vesicles in aqueous solution with the mixture of simple, commercially available, single tailed cationic (cetyl trimethylammonium tosylate, CTAT) and anionic (sodium dodecyl sulfate, SDS) surfactant.9 The size, surface charge and permeability of the vesicles can be finely adjusted by varying the surfactant ratio. The formation of vesicle is due to the production of IPA which acts as double chained zwitterionic surfactant. Compared to conventional vesicles which are prepared by mechanical disruption or insoluble liquid crystalline dispersions these vesicles are much stable and vesicles are spontaneously formed immediately after combining the two oppositely charged surfactant. The vesicles are characterized by quasi-elastic light scattering (QLS), freeze fracture TEM and glucose entrapment experiment. The size of vesicles is varied in between 30-80 nm. The ion pairs have geometrical feature of small head group and long tail group which is necessary for the vesicle formation and the residual surfactants help to fluidize the membrane. The result obtained from SDS-CTAT mixture is the representative of other surfactant mixtures. A substantial effort has been made to investigate the structural details of the mixture of cationic/anionic/non-ionic surfactants using cryo-TEM and small angle neutron scattering (SANS) measurements and it is mainly focused on the formation of SUVs in the isotropic part of the phase diagram.64-68Both the measurements confirm the simultaneous presence of open and closed bilayer structure of SOS (sodium octyl sulfate) and CTAB (cetyl trimethylammonium bromide) mixture. Further, Grillo et al. investigated the effect of salt on the self-assembly in SOS rich mixture of SOS and CTAB.65In presence of salt, sodium bromide (NaBr), the micelle become much larger at the transition between micelle and vesicle. With increasing concentration of NaBr, the morphology of the vesicles is also changed according to the following sequence: vesicle → disks → perfolated vesicle, as evidenced by SANS measurements. Mixing of cationic/anionic surfactant can weaken the electrostatic interactions between the charged head groups which promote micelle growth and finally form wormlike micelles.56,69 The formation of 13 ACS Paragon Plus Environment

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wormlike micelle can be justified by the non-monotonous change in the rheological properties at high surfactant concentration.56Wang et al. demonstrated the micelle to vesicle transition in the mixture of anionic sulfonate Gemini surfactant and CTAB at pH 9.5 using Cryo-TEM, DLS, turbidity, rheology and isothermal microcalorimetry (ITC) measurements.70They have observed reversible transition between micelle, wormlike micelle and vesicle after mixing the two surfactants in different ratios. The wormlike micelle shows higher viscosity and shear thinning property due to the strong hydrogen bond, complexation, and conjugation between the surfactants. Further, the vesicular aggregate does not precipitate even at high concentration of surfactant and 1:1 surfactant ratio which suggests that gemini surfactant could be an effective approach to improve the solubility of cationic/anionic surfactant mixture. During the transition, hydrophobic interaction between the hydrocarbon chains, hydration energy as well as deionization of head groups play a significant role. However, the origin of the transition is from the variation of the electrostatic interactions between the head group of the surfactants. In recent times, researchers are interested to investigate the formation of vesicles in room temperature ionic liquids (RTILs) or using RTILs. RTILs are low melting organic salts which show unique physiochemical properties including thermal stability, broad liquid range, low vapor pressure etc. The solvophobic interactions between the IL and surfactant molecules play a crucial role. IL with long hydrophobic alkyl chain shows surface active properties and they are termed as SAILs (surface active ionic liquids). It is well established that the surface active property and directional polarizability of SAILs help to prepared different self-organized assemblies which can exhibit improve templating behavior for the synthesis of nanoparticles.71,72 Besides, the anionic and cationic constituents of SAILs can be easily modified according to the desired properties.73,74Recently, Wang et al. have demonstrated concentration dependent micelle to vesicle transition of 1-alkyl-3-methylimidazolium bromides, [Cnmim]Br (n=10,12,14) in aqueous solution.72 Further, these SAILs can form vesicular assemblies in presence of other assemblies or negatively charged surfactants.59,75Recently, Dutta et al. investigate the micelle-vesicle-micelle transition of anionic surfactant in presence of SAILs (C12mimbr and C16mimBr) using zeta potential, TEM, DLS and turbidity measurements.59 Zeta potential measurements suggest that incorporation efficiency of C16mimBr in SDBS micelle is much better than C12mimBr due to the strong hydrophobic as well as electrostatic interaction between the two molecules. Further, Ghosh et al. also investigated the micelle-vesicle-micelle 14 ACS Paragon Plus Environment

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transition of cationic CTAB and anionic surfactant like IL, 1-butyl-3-methylimidazolium octyl sulfate, [C4mim][C8SO4] (figure 3).56 Depending on their relative amount, these surfactants either mixed together to form vesicles or mixed micelles. [C4mim][C8SO4] forms spherical micelle in aqueous solution (~3nm). At low volume fraction of CTAB (

)

transparent, low viscous micellar phase was observed and this region was composed of pure micelle or mixed micelle (figure 3(a)). Further increase in the concentration of CTAB leads to the formation of vesicular aggregates and the solution was turned into blue in colour and a white precipitate was obtained which was due to the formation of larger vesicular aggregates (figure 3(b)). At higher molar ratio, (

=0.80) an ambiguous phase evaluation was observed which

was clear but viscous in nature and it signifies the formation of elongated micelle. At mole fraction higher than 0.80 of CTAB, clear micellar phase was observed. The different phases in different fraction of CTAB in the mixture were successfully characterized by turbidity, zeta potential, surface tension, conductivity, DLS, TEM measurements and steady-state anisotropy measurements (figure 3). Beside the oppositely charged surfactants, small amphiphiles can also induce the formation of vesicles in aqueous solution and one of the most well studied amphiphiles are cholesterol, 5methyl salicylic acid (5mS) etc. The role of cholesterol on the vesicle formation is discussed above.54,75 Sarkar and coworkers have investigated the formation of vesicular aggregates of differently charged surfactant molecules, such as SDS, CTAB, SAILs etc in presence of cholesterol.58,75 The aggregation of surfactant molecules is depended on their packing parameter. Salicylic anion can strongly bind to the surfactant head group and the phenyl group of the salicylic anion can also be embedded into the hydrophobic portion of the surfactant.76 Therefore, it leads to the increase in the volume of the micelle core. Thus, addition of 5mS into the micellar solution reduces the effective head group area and increase the effective cross sectional area of hydrophobic chain and with increasing concentration of 5mS transition of spherical micelle to elongated micelle with bilayer structure is observed (figure 4(a)). Raghavan et al. observed the temperature dependent vesicle to wormlike micelle transition of CTAB/5mS mixture using SANS and rheology measurements.76The vesicle to micelle transition causes the solution to switch from the low viscous, Newtonian fluid to viscoelastic, shear thinning fluid. Vesicular aggregates are formed in presence of excess 5mS molecules and some of the 5mS molecules are


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weakly adsorbed at the aggregate interface. Therefore, they have the tendency to desorb upon heating and this induces the change in the molecular geometry from vesicle to wormlike micelle.

Figure 3. (a) Pictorial Representation of Oscillation between Micelle-Vesicle-Micelle in aqueous [C4mim][C8SO4] solution with Addition of Different Volume Fraction of CTAB. (b) Phase behavior (optical micrographs) of the aqueous [C4mim][C8SO4]-CTAB with increasing volume fraction of CTAB. (c) TEM images of vesicles and elongated micelles composed of [C4mim][C8SO4] and CTAB. (d,e,f,g,h) Viscosity, Surface tension, Conductivity, Size (determined by DLS) and Zeta potential [C4mim][C8SO4]/CTAB solution at different volume fraction values of CTAB. Reproduced with permission from ref. 56. (Copyright 2013, American Chemical Society). 5mS induced vesicle formation is also studied by Roy et al in presence of C16mimCl.63 With the addition of 5mS into the solution, the micellar aggregates are transformed into elongated micelle and finally vesicle (figure 4(b)). Besides, they have also observed temperature dependent reversible transition between C16mimCl/5mS vesicular aggregates and C16mimCl/5mS micelle. The transformation is confirmed by the turbidity measurements and DLS measurements. Besides amphiphilic surfactants, non-ionic triblock copolymer micelle can also form vesicles in presence 16 ACS Paragon Plus Environment

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of 5-mS.48Beside 5ms, DNA nucleotide AMP (adenosine -5/-monophosphate) also induces the formation of larger micellar aggregates composed of cationic (C12mimCl) and anionic ([C4mim][C8SO4]) surfactants (figure 4(c)).77However, the insertion of negatively charged AMP into the cationic surfactant is more prominent compared to that of anionic surfactants. Therefore, the tunable micelle-vesicle transition and concomitant viscosity increase upon heating have a utility in a range of areas, including microfluidics, controlled release, and oil recovery.76 The surface properties of SAILs can be tuned by carefully modified the cationic as well as anionic constituents. Pyne et al. have successfully synthesized L-glycine amino acid derived cholesterol based SAIL which can form microemulsion in non-aqueous environment and vesicle in aqueous medium.78 Moreover, Banerjee et al. prepared giant vesicles using SAILs. In this case, the SAILs were synthesized by replacing the cationic part of Aerosol OT (AOT) with cations having alkyl chain of different lengths, CnmimCl (n = 8,12,16).73 The size of the vesicles was depended on the chain length of alkyl group. The vesicles were characterized by the FLIM (fluorescence lifetime imaging microscopy) and TEM measurements. Further, they have studied the conformational dynamics of BSA protein inside these vesicular aggregates.


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Figure 4. (a) Schematic representation of the temperature dependent vesicle-to-wormlike micelle transition in CTAB/5mS mixtures. (b) Schematic representation of concentration dependent micelle-elongated micelle-vesicle transition in C16mimCl/5mS mixtures and (c) Schematic representation of formation of different aggregates in SAIL/ AMP mixtures. Image (a) is reproduced with permission from ref. 76. (Copyright 2006, American Chemical Society). Image (b) and (c) are reproduced from ref. 63 (Copyright 2016, American Chemical Society) and ref. 77 (Copyright 2016, American Chemical Society). 3. Excited State Dynamics in Vesicles. 3.1. Solvation Dynamics and Rotational Diffusion in Vesicles. Water molecules present at the hydration layer of the self-assembled systems play a significant role in controlling the structure, reactivity and heterogeneity of the system and the behavior of this confined water is very different with respect to polarity, viscosity and pH compared to that of bulk water.79Since vesicle can mimic the complex biological membrane in a much simpler form, study of water dynamics near the hydration layer of vesicles has gained a significant attention in last few years.80-82Water dynamics at the lipid/water interface has been extensively studied by vibrational sum frequency generation, femtosecond mid-IR pump-probe and terahertz (THz) spectroscopy and it is well established that different types of water molecules exist at the zwitterionic lipid/water interface: (1) phosphate bound water, (2) choline bound water and (3) hydrophobic water.81Zhao et al. have investigated the dynamics of water at the surface of multibilayer phospholipid, DLPC probed with ultrafast vibrational pump-probe spectroscopy and showed that the water molecules near the bilayer are distinct in nature compared to the hydration water at the phosphate and choline group.80 They have also observed two different populations of water molecules in the vesicle. Based on this, they have suggested that one component might be a clathrate-like water cluster near the hydrophobic choline groups and another component might be related to the water molecules near the phosphate groups. Further, Kundu et al. showed that the dynamics and structure of water molecules between DMPC vesicles change dramatically as the structure of the membrane is changed from gel phase to liquid crystalline phase.81Recently, Yamada et al. using quasi-elastic neutron scattering (QENS) study, have also proposed that hydration water in DMPC vesicles can be categorized into three types of water: (1) free water molecules whose behavior is slightly different from the bulk water, (2) loosely bound water, whose dynamics is 1order of magnitude slower than the free water and (3) tightly bound water (figure 5(a)).82 The number of free water molecules remains constant as they vary the 18 ACS Paragon Plus Environment

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temperature of the system. However, the number of loosely bound and tightly bound water changes by varying the temperature. Plasma membrane contains a wide variety of proteins and biomolecules which are essential for the cell functions and these membranes are frequently modeled with different planar phospholipid bilayers and vesicles. The curvature of the vesicles influences different structural and thermodynamic properties of vesicle. In this aspect, recently, Fayer et al. have also performed the size dependent solvation dynamics inside phospholipid vesicles using 2D IR vibrational echo spectroscopy83 and they have concluded that the as the size of the vesicle decreases the interior structural dynamics of the membrane become faster and as the size of the vesicle increases the interior dynamics approach towards that of the planar bilayer. Beside these techniques, solvation dynamics using femtosecond upconversion and time correlated single photon counting techniques has been extensively studied as a sensitive method for better understanding the hydration behavior in the vesicular aggregates.84,85For this, the emission decays of fluorophore molecule are collected at several emission wavelengths and then, from the parameters of best fit to the fluorescence decays, TRES (time resolved emission spectra) is constructed following the method of Fleming and Marencolli.86 After fitting the each TRES with log-normal function, the peak frequency of the function is calculated and from this, solvent correlation function, C(t) is generated which is defined by (1) In the above equation,

,

and

are the peak frequency at time t, 0 and infinity.

Further, the C(t) is fitted with a multi-exponential function to obtain the solvation time. Bhattacharyya et al. have performed the solvation dynamics of Coumarin-480 (C-480) in DMPC lipid (figure 5(b)).85 Using the femtosecond upconversion technique, they have first reported the ultrafast component of 1.5 ps for C-480 in DMPC. Due to the microheterogeneity of the vesicle, they have also observed significant red edge excitation shift (REES) after exciting C-480 at two different excitation wavelengths (390 nm and 430 nm). In DMPC, C-480 molecules are located in different region with varying static and dynamic electrostatic responses (figure 5(b)). At , the probe molecules which are located at the restricted region inside the lipid vesicles are excited. However, for

=430 nm, the major contribution of the total emission is

due to the probe molecule in highly mobile environment (water pool of the vesicle). Therefore, at 19 ACS Paragon Plus Environment

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the solvation time was found to be 1.5 ps. However, at

two

substantially slower components of 250 and 2500 ps was observed. In the DMPC vesicle, water molecules near or inside the membrane bilayer are hydrogen bonded to the surfactant molecules. These water molecules are immobilized and would give a slow solvation time. However, water molecules at a distance greater than 3-4 A0 from the lipid head group are behave like bulk water. Thus, the timescale of solvation dynamics varies sharply with the distance from the lipid bilayer. The ultraslow component which was observed at

, may arise from number of

sources. The main polar species responsible for the solvation dynamics is the water molecules as the dynamics of other polar moieties are very slow compared to that of water. In this aspect, Nandi and Bagchi proposed that in biological systems, slow component in solvation dynamics arise due to the dynamic exchange between the bound and free water molecules and the rate determining step of the solvation is the rupture of hydrogen bond between the water and membrane.79Moreover, relaxation of the water molecules which are attached to the surfactant head groups require rupture of hydrogen bond along with the coherent motion of the surfactant chain and the timescale of such motion is quite long and it may be the possible reason of such slow component. Another origin of the ultraslow component is the diffusion of polar probe molecule from non-polar region to more polar region in the excited state which leads to spectral narrowing. Roy et al. further performed the solvation dynamics in DMPC vesicle in presence of different sugar molecules and they have used two different fluorophores (C-153 and C-480) which are located in different region of the vesicle depending on their hydrophobicity.54Sugar molecules intercalate the phospholipid head group with the hydrogen bonding interactions and replace the small sized water molecules from the membrane surface. C-153, being more hydrophobic fluorophore is located at the bilayer region of the vesicle. However, C-480 is located near the water pool region of the vesicle. In both the cases, they have observed increase in average solvation time with addition of different sugar molecules. However, the increase in solvation time is much higher in sucrose compared to that of other sugars, such as trehalose and maltose. Recent simulation studies also suggests that the extent of hydrogen bonding between sucrose and phospholipid head groups is much higher compared to that of other sugar molecules.87Compared to C-153, more change in solvation time is observed for C-480. C-480 molecules are mainly localized at the head group region of the vesicles and the effect of sugars is maximum at the head group region. Besides the lipid vesicle, solvation dynamics has also been 20 ACS Paragon Plus Environment

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performed in other vesicles, such as niosomes, SAIL based vesicles etc. by several groups50, 75

and in every cases, multimodal solvation time has been observed which can be explained by the

Nandi and Bagchi model as described above. Beside the ensemble average measurements, Bhattacharyya et al. first performed the solvation dynamics of a giant single lipid vesicle (~20

) under a microscope.88 They have attempted to understand the solvation dynamics in

different region of the vesicle using single molecule spectroscopy (figure 5(c)). They concluded that solvation time within a vesicle is not same everywhere and it depends on the position as well as microenvironment of the probe molecule which signifies the dynamic microheterogeneity within a single vesicle.

Figure 5. (a) Schematic presentation of dynamic behavior of hydration water molecules between phospholipid vesicles using QENS measurements. (b) Solvation dynamics in a single vesicles C(t) is plotted in different region of a single vesicle. The size of the vesicle is approximately 20 µm and the different regions are shown by the white circle (c) Schematic representation of different region of DMPC vesicle; femtosecond fluorescence decay curves of C-480 at different emission wavelengths and decay of solvent correlation function, C(t) at three different excitation wavelengths (solvation time become faster after exciting at the red end of excitation wavelengths). Image (a) is reproduced from ref. 82 (Copyright 2017, American Chemical Society). Image (b) is reproduced from ref. 88 (Copyright 2012,


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American Chemical Society) and Image (c) is reproduced from ref. 85 (Copyright 2006, Wiley). Beside the solvation dynamics, the rigidity and hydrophobicity of the membrane can be determined using time resolved anisotropy measurements. The rotational diffusion of the probe molecules is depended on their location as well as the surface charge of the membrane.58,59 The nature of organized assemblies determines the location of ionic or hydrophobic fluorophores inside the aggregates. Therefore, proper choice of probe molecule and its rotational dynamics is very important to understand the hydration behavior and the rigidity of the assemblies. Ghosh et al demonstrated that effect of surface charge of ionic surfactant (SDS, CTAB) and cholesterol forming vesicles on the translational and rotational diffusion cationic probe molecule, Rhodamine 6G (R6g).58In cholesterol-SDS vesicles, due to the strong electrostatic attraction between the R6g and head group of the vesicle, the rotational relaxation became slower compared to SDS micelle. However, in case of CTAB-cholesterol aggregates, due to the enhanced hydrophobicity and electrostatic repulsion, R6g molecules migrated from vesicle bilayer to water. Thus, the rotational relaxation time became faster in vesicle. Besides the surface charge, rigidity of the membrane also determines the rotational relaxation of the probe molecule. Dutt et al have used time resolved anisotropy as a tool to determine the rigidity of vesicles composed of C16mimCl/cholesterol and BHDC (benzenedimethylhexadecylammonium chloride)/cholesterol using C-153 as a probe molecule.59C16mimCl and BHDC both form micellar aggregate in aqueous solution. In presence of cholesterol, the rotational relaxation time of C-153 in both the cases significantly increased. Therefore, it signifies the transformation of micellar structure into vesicular aggregates. Due to the poor water solubility and strong hydrophobicity of C-153, it is mainly located at the bilayer of the vesicle and the slower rotational relaxation of C-153 in C16mimCl/cholesterol vesicle compared to that of BHDC/cholesterol vesicle signify that the bilayer of the former vesicle is much rigid compared to the later one. 3.2. The dynamic properties of vesicles studied by fluorescence resonance energy transfer (FRET). Over the last few decades, FRET has been used as potential spectroscopic tool for the determination of distance in numerous biological macromolecules, such as protein, DNA and 22 ACS Paragon Plus Environment

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biopolymers. According to the Forster theory, the rate of FRET is depended on the distance between donor (D) and acceptor (A) and the relative orientation of the transition dipole moment. Each D-A pair has a characteristics Forster distance which determines the distance limit accessible to FRET measurements. Bhattacharyya et al. showed that the standard Forster theory can be applied to estimate the D-A distance when they are separated by surfactant chains in the aggregates.89According to the Forster theory, the rate of FRET can be determined by the following equation (2) In the above equation,

is the rise time of the A molecule in presence of D.

of D in absence of A and R is the distance between the D and A. distance at which the rate of energy transfer is 50%.

is the lifetime

, the Forster distance is the

can be calculated by the following

equation (3) Where

is the refractive index of the medium,

orientation factor and

is the quantum yield of the D,

is the

is the spectral overlap between the emission spectrum of D and

absorption spectrum of A. Mandal et al. have used FRET as a tool to monitor the micelle-vesicle transition in an aqueous mixed SAIL system (figure 6).90 The vesicle was composed of SAILs, C12mimCl and [C4mim][C8SO4]. C-153 was used as a D and R6g was used as A. R6g is preferentially located at the polar water pool of the vesicle and C-153 is located at the hydrophobic bilayer of the vesicle (figure 6(a)). As FRET is strongly depended on the D-A distance, the structural transition from micelle-vesicle can be monitored by FRET. In [C4mim][C8SO4] micelle, an ultrafast component of 3.3 ps was observed which correspond to a D-A distance of about 15 A0. However, in SUV composed of [C4mim][C8SO4]/ C12mimCl, FRET occurred on multiple timescales of about 250 and 2100 ps, which correspond to the D-A of about 33 and 47 A0(figure 6(c)). The multiple timescales also signifies the dynamics heterogeneity of the vesicles. The authors have also estimated the bilayer thickness from the cryo-TEM images which was closed to 50-60 A0 (figure 23 ACS Paragon Plus Environment

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6(b)). Thus, they have concluded that the multiple timescales of FRET solely came from the contribution of intravesicular FRET. Besides, the common surfactant containing vesicles (CTAB/SDS), the thickness of the bilayer was reported to be in the range of 24-34 A0 and distance reported by the FRET measurements by Das et al. in this vesicular system was also correlated well with the bilayer thickness.91 Therefore, the D-A distance obtained from FRET measurements can provide useful information regarding the average thickness of bilayer. Recently Roy et al. have also performed the excitation wavelength dependent FRET on the niosome composed of non-ionic triblock copolymer, F127/cholesterol using C-153 as a D and R6g as A.53 Due to the dynamic heterogeneity of the niosomes, they have also observed multiple timescales on FRET parameters (figure 2). Besides, the contribution of faster rise component increased significantly after varying the excitation wavelength from 408 nm to 440 nm. These results also suggest that the FRET is completely intravesicular in nature.

Figure 6. (a) Schematic representation of micelle to vesicle transition of SAILs and their effect on FRET parameters (b) Cryo-TEM images of mixed SAILs [C4mim][C8SO4]/[C12mim]Cl vesicles (xC12mimCl = 0.50). (Red arrows indicate the bilayer of the vesicles) and (c) Picosecond time-resolved fluorescence decays of acceptor R6g in the absence and presence of C153in SAILs forming different microenvironments ofmixed micelles (x[C12mim]Cl = 0.25) and mixed SAILs vesicles (x[C12mim]Cl = 0.50). (λex = 408 nm, λem = 570 nm). Images are reproduced from ref. 90. (copyright 2014, Wiley).


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Lipid bilayer shows an ordered to disordered transition at a particular temperature range which is known as transition temperature (

) which is 23.90 for DMPC lipid. Thus, below this

temperature lipids are in solid phase with reduced fluidity and above this temperature they are in liquid phase with greater fluidity. In this regard, Ghatak et al. have also studied of effect of membrane fluidity of DMPC vesicles on FRET parameters below and above transition temperature.92Above the

the FRET efficiency between C-153 and R6g is increased due to the

greater solubilization at liquid crystalline phase. Thus, the diffusion of the dyes through the lipid bilayer was increased which in turn the surface density of acceptor is increased. Beside the ensemble average measurements, recently single molecule FRET (smFRET) has also been employed to determine the SNARE mediated vesicle fusion kinetics.93 Different FRET based lipid mixing and content mixing assays are used to exploit the different substages of fusion, such as docking, hemifusion, pore expansion and full fusion. The exact time scale of individual step can be determined by smFRET measurements. In last few years, several research articles have been dedicated to understand the membrane fusion process using smFRET measurements. However, an exhaustive discussion regarding this is beyond the scope of this present article. 3.3. Application of Fluorescence Correlation Spectroscopy (FCS) in Vesicle study. FCS technique is based on the statistical analysis of fluorescence intensity fluctuations detected from a fluorophore in a very small volume. Nowadays, FCS is a commonly used tool to efficiently determine the local concentration, translational as well as rotational diffusion, reaction constants and molecular aggregation etc. Further FCS can be used to investigate the diffusion properties of membrane component. Therefore, it is an important tool to study the membrane structure and biological reactivity.94 In FCS, diffusion of the fluorophores through the confocal volume and/or different photophysical processes cause fluctuation in the confocal volume. The fluctuation is further analyzed by function which is defined by (4) In the above equation,

is fluctuation of fluorescence intensity at time t,

is the lag time

and is the average fluorescence intensity. The experimental autocorrelation function is then fitted with a mathematical model to extract different parameters, such as diffusion 25 ACS Paragon Plus Environment

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coefficient, concentration etc. The detection volume is usually considered as a 3D Gaussian profile and the correlation function can be defined by the following equation (5) where,

is the diffusion time of the fluorophore within the confocal volume, S is denoted as the

structure parameter of the excitation volume. The proper choice of fluorophores for the successful FCS measurement is essential and the fluorophore must have high quantum efficiency, high photostability, large absorption cross section and considerable partition into the lipid bilayer. Different long alkyl-carbocyanine dyes, such as DiD, DiA, Dio are widely used as a fluorescent membrane component. They are mainly characterized by their well defined transition dipole moment which is parallel to the plane of membrane and lipid moieties containing acyl chain of different length and different degree of saturation. Besides, to monitor the packaging and membrane fluidity using FCS another ploymethine dye, merocyanine-540 (MC540) can be used.95 MC540 binds to the outer leaflet of the membrane and undergoes trans-cis isomerization after photoexcitation which can be monitored by the FCS in a straightforward manner. The isomerization kinetics is sensitive to the membrane viscosity and temperature. Using FCS, transition to long lived weakly fluorescent state can be monitored via the fluctuation in the fluorescence intensity from low number fluorophores. Thus, in this case, the fluctuation in the fluorescence intensity is generated from the translational diffusion as well as the isomerization and back-isomerization of MC540 and the autocorrelation function can be defined by (6) In the above equation,

is the average fraction of fluorophores within the confocal volume

being in a non-fluorescnet cis isomer form and

the relaxation time related to trans-cis

isomerization process. Chmyrov et al. have investigated the influence of membrane curvature, membrane polarity and cholesterol content in DOPC vesicles using FCS measurements (figure 7(a)).95They did not observe any influence of the membrane curvature and polarity on the isomerization rate. However, rate of isomerization was decreased with increasing concentration of cholesterol of membrane and isomerization kinetics directly reflects the lipid head group


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viscosity. Thus, the trans-cis isomerization kinetics of MC540 using FCS can be a powerful parameter to monitor the membrane dynamics. Determination of absolute diffusion coefficient from FCS relies on the exact shape and size of the confocal volume which is often considered as a 3D Gaussian shape. In literature, different calibration free FCS techniques are reported over the past few decades and among them, most popular calibration free technique is Z scan FCS which uses multiple focus plane to determine the diffusion coefficient in supported phospholipid bilayers (SPBs).96 In this approach, the correlation curve is collected at each axial sample position and analyzed with the appropriate planar diffusion model. The diffusion time and particle number are then separately plotted as a function of relative sample position and it yields parabolic dependencies and from this, the calibration-free diffusion coefficient can be obtained.

Figure 7. (a) Cis-trans isomerization of MC-540 after photoexcitation, (b) Normalized FCS curves recorded from MC-540 associated with DOPC liposomes with different cholesterol concentrations. (Inset: kISO and σBISO versus cholesterol concentration in the liposomes.) (c) FCS traces of DCM in water, oleate ethylamine (OEA) vesicle and oleic acid/oleate vesicle. Distribution of Dt values obtained by performing the FCS experiments more than 10 times 27 ACS Paragon Plus Environment

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and the mean value of Dt were determined by fitting the distribution to Gaussian function. Images (a) and (b) are reproduced from ref. 95 (copyright 2015, American Chemical Society) and image (c) is reproduced from ref. 42 (copyright 2017, American Chemical Society) Mojumdar et al. have performed the FCS measurements in different region of 20

sized

vesicles and they have observed significant variation of the diffusion coefficient in different region of vesicle which clearly indicates the microheterogeneity of single vesicle.88The microheterogeneity of fatty acid based vesicles is also showed by Kundu et al. using FCS measurements and it is shown in figure 7(b).42They have also investigated the change in the translation diffusion parameter of the fluorophore during micelle-vesicle transition.58 Similar to the rotational relaxation, the translation diffusion of the fluorophore is also depended on the charge, hydrophobicity and rigidity of the vesicle. Dey and Ghosh et al. have also studied the diffusion behavior of fluorophores in gel phase and fluid phase of lipid vesicle.97,98 As diffusion is sensitive to the viscosity of the medium, the diffusion time in fluid phase is much faster compared to that of gel phase. Thus, FCS based measurements provide significant information regarding the membrane dynamics and organization. Although FCS measurements on lipid membranes are prone to several experimental difficulties recent technical developments have allowed successful application of FCS to more complex systems, such as cellular membranes. 4. Self-Assembly of Single Amino Acids into Fibrils and their Interaction with Vesicles. Fibrils are the thermodynamically most stable conformations of different proteins and peptides in water and they are termed as “amyloid” when they are found in pathological deposits in human tissue. Different degenerative disorders, such as Alzheimer’s disease, parkinson’s disease and type II diabetes are associated with the formation of amyloid fibrils and toxicity of the assemblies is generally related to the properties of the well-formed fibrilar structure.13-15 However, the early forming oligomers of amyloid aggregates have an inherent toxicity to cell. Therefore, detection of early step of protein aggregation is important to prevent the formation of such disorders. Recently, it is recently found that not only polypeptides or proteins but also single amino acids such as L-Phe, L-Tyr can form well-ordered amyloid like fibrilar structures.16,17,99,100 These assemblies can also bind to the amyloid markers, such as congo red, Th T (Thioflavin-T) etc. and these assemblies have significant cytotoxicity similar to amyloid fibrils.17 Single crystal X-ray diffraction analysis demonstrates the tight packing of the L-Phe 28 ACS Paragon Plus Environment

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which resembles β sheet like structure (figure 8c). Figures 8 (a) (i) to (iii) represent the TEM, SEM and confocal microscopy images of L-Phe fibril reported by Gazit and coworkers.16 The fibrillar assembly of L-Tyr was shown by Perween et al. (figure 8 a(iv)).100 Banik et al. further provide the FLIM image of L-Phe and L-Tyr fibrils using DCM (dicynomethylene-4H-pyran) as a dye (figure 8b).101 ThT is well known for the detection of amyloid assemblies.102 However, it is reported that ThT facilitates the amyloid fibril formation.103In this regard, Cheng et al. have shown that DCM is a good probe for in vivo cerebral imaging of amyloid-β fibrils.104 The cytotoxicity of the L-Phe fibrils may provide an explanation for the mental destabilization observed in PKU patients and the detection of L-Phe assemblies in the brain of PKU model mice also support this notion. Amyloid formation is dependent on various factors, such as chirality of the molecule, pH of the medium, nature of solvent, sequence of amino acid within the peptide etc.105,106 It is very much important to discuss the inhibition strategies of amyloid fibrils. It has been observed that polyphenols can effectively inhibit the amyloid formation.107 Small molecules, cucurbit[7]uril and polycyclic compounds, such as crown ethers can also arrest the amyloid formation.108-111 Sarkar et al. investigate the fibril formation mechanism of L- Phe and L-Tyr in detail and their results indicate that the fibril formation mechanism is completely different for these two amino acids.101 For L-Phe, -NH3+ and CO2- of two neighboring molecules interact via hydrogen bonding interaction and polar interaction. However, in case of L-Tyr, NH3+ has no role in the fibril formation. Therefore, the hydrogen bonding partners for L-Tyr fibril are the –OH and CO2-. Accumulation of high concentration of Phe into the brain causes brain damage and this neurodegenerative disease is known as PKU which is already mentioned in the earlier section. Adler-Abramovich et al. have demonstrated that Phe can also self-assembled into fibrils in pathological condition and these fibrillar assemblies show significant cytotoxicity.16 Therefore, understanding the action of these self-aggregated fibrils on the cell membrane is crucial for developing new therapeutic approaches for the metabolic disorders, such as PKU and more easier approach to investigate this has been the use of simplified systems with model phospholipid membranes and single amino acids or short sequences of amino acids.


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Figure 8. (a) (i) TEM (scale bar is 1 µm) (ii) SEM (scale bar is 20 µm) (iii) Thioflavin T (ThT) stained confocal microscopy images of L-Phe fibrils (scale bar is 10 µm) (iv) SEM image of L-Tyr fibril (scale bar is 2 µm); (b) FLIM images of L-Phe (i, scale bar 100 µm) and L-Tyr (iii, scale bar 100 µm) fibrils and FESEM images of L-Phe (ii, scale bar 4 µm) and L-Tyr (iv, scale bar 100 µm) fibrils. (c) Amyloid like self-assembled behavior of L-Phe observed by electron microscopy and binding to amyloid specific dye molecules, such as congo red, Th T. Crystal structure of L-Phe is regarded as supramolecular strand like organization. These assemblies are cytotoxic and their deposition can be observed in the brain of PKU patients. The self-assemblies can be inhibited by D-Phe isomers and by depletion with specific antibodies raised toward the supramolecular assemblies. Images (a) [(i), (ii) and (iii)] are reproduced from ref. 16 (Copyright 2012, Nature Publishing Group), (a) [(iv)] is reproduced from ref. 100 (Copyright 2013, Royal Society of Chemistry), image (b) is reproduced from ref.101 (Copyright 2017, American Chemical Society) and image (c) 30 ACS Paragon Plus Environment

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is reproduced from ref. 17 (Copyright 2015, American Association for the Advancement of Science). Perkins et al. have claimed that Phe does not form any aggregated structure in solution.19 However, the aggregation occurs at interface and large change in the morphology of the DPPC monolayer was observed at air-water interface in presence of L-Phe.112 With the use of molecular dynamics simulation and different experimental techniques it has been observed that L-Phe intercalates into the DPPC film at air-water interface (figure 9a) and it affects the surface tension, phase morphology and ordering of the DPPC film.112 Moreover, Phe increases the membrane permeability of the DPPC membrane.19 However, no change in the shape or size of the vesicle was observed in Cryo-TEM measurements in presence of Phe (figure 9b) and no traces of Phe aggregate was observed in the TEM images. In this regard, MD simulation study suggests that small clusters of Phe form small pores in lipid membrane.112 Thus, the change in permeability in presence of Phe is either due to the large-scale change in the morphology of DPPC vesicles or a small scale change around the Phe clusters. Consequently, the increase in the permeability is the primary reason behind the damage associated with untreated PKU and these results are very consistent with the earlier observation which stated that decreasing the Phe serum level can reverse the brain abnormalities in adults.

Figure 9. (a) Schematic representation of Phe intercalation into the lipid interface. The red arrow indicates the rotation of carboxylate group as a previous step before the insertion of Phe into the membrane. (b) Cryo-TEM images of DPPC vesicles treated with (left) and without (right) Phe. Image (a) is reproduced from ref.113 (Copyright 2015, American Chemical Society) and image (b) is reproduced from ref. 19 (Copyright 2017, American Chemical Society).


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Rosa et al. recently studied the interaction between Phe and DPPC model membrane using ATRFTIR spectroscopy and their results suggest that phenyl ring of Phe interact hydrophobically with the membrane. Further, dipole interaction and hydrogen bonding interaction between –NH+ group and water phase also stabilize the DPPC-Phe conjugates. Based on the results, Perkins et al. have further assumed that useful interactions of single amino acids with membrane can set a direct path from amino acid monomers to functional proteins without any need for genetic encoding.19 Moreover, it can also relate to the origin of life. According to the fluid mosaic model membrane proteins are the essential component of modern cell membrane and the simple amino acids has the ability to perform some functions of membrane proteins which help to bridge the gap between living and non-living organism. The restricted water pool in the vesicle can promote the non-enzymatic peptide bond formation. Thus, this system is particularly advantageous in this regard and needs much more attention. Conclusions and Outlook In this feature article, we have revisited the self-assembly of different amphiphilic molecules which form vesicles and fibrils. The formation of phospholipid based vesicles, fatty acid vesicles and ionic as well as non-ionic surfactant based vesicle have been discussed. Further, ionic surfactants which form micellar aggregates in aqueous medium can be transformed into more complex structures, such as wormlike micelle, vesicles etc. in presence of other oppositely charged surfactants, amphiphiles (cholesterol, 5mS etc). The change in the geometry of the aggregates is monitored using light scattering, viscosity, conductivity, surface tension and electron microscopy measurements. The water dynamics in vesicle is complex in nature and much slower compared to that of bulk water. These behaviors in confined assemblies mimic superbly different biological and chemical processes occurring in nature. Different spectroscopic studies also suggest the existence of different types of water molecules inside vesicle and the solvation time is varied from ns to ps region due to the dynamic exchange between bound water and free water. The dynamic heterogeneity of the vesicle is also probed by FRET and FCS measurements. FCS measurements also determine the change in the membrane fluidity in presence of different amphiphiles. Thus, the dynamics of the vesicular aggregates is important for the complete understanding of the organization, reactivity and heterogeneity. Further, amino acids based amphiphiles also self-assembled to form fibril in aqueous medium and the formation 32 ACS Paragon Plus Environment

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of such aggregates is governed by the strong non-covalent

interaction and hydrogen

bonding interaction. The fiber like network is characterized by the FESEM and FLIM measurements. Beside the mentioned amino acids, other amino acids and the nucleic acid bases can also form different supramolecular aggregates and they are under investigation. Further, delivery of these biomolecules through vesicle can also modulate the kinetics of the fibril formation. The membrane and single amino acids interaction is of particularly important to understand the origin of the different metabolic disorder caused by the aggregation of these amino acids. Finally, a complete understanding of such processes hold promise for better understanding of human diseases and physiology and covers the way to improved diagnostics, drug development and drug delivery. Acknowledgment: N.S. gratefully acknowledges SERB(Grant No: IR/S1/LU-001/2013 dated 24/03/2015), Department of Science and Technology (DST), Council of Scientific and Industrial Research (CSIR), Government of India for providing generous research grants. N.K. and D.B. are thankful to IIT Kharagpur for their research fellowships.

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(90) Mandal, S.; Kuchlyan, J.;Banik, D.;Ghosh, S.;Banerjee, C.;Khorwal, V.; Sarkar, N. Spontaneous Micelle-to-Vesicle Transition in Aqueous Mixed Surface Active Ionic Liquid System: Characterization and Insight from Ultrafast Fluorescence Resonance Energy Transfer. Chem. Phys. Chem. 2014, 15, 3544. (91) Das, A. K.; Mondal, T.; Sasmal, D. K.; Bhattacharyya, K. Femtosecond Study of Ultrafast Fluorescence Resonance Energy Transfer in a Catanionic Vesicle J. Chem. Phys. 2011,135, 074507. (92) Ghatak, C.; Rao, V.G.; Pramanik, R.; Sarkar, S.; Sarkar, N. The Effect of Membrane Fluidity on FRET Parameters: an Energy Transfer Study inside Small Unilamellar Vesicle. Phys. Chem. Chem. Phys., 2011, 13, 3711–3720. (93) Yoon, T.Y.; Okumus, B.; Zhang, F.; Shin, Y.K.; Ha, T. Multiple Intermediates in SNARE-induced Membrane Fusion. Proc Natl Acad Sci U S A 2006, 103, 19731-19736. (94) Chiantia, S.;, Ries, J.; Schwille, P. Fluorescence Correlation Spectroscopy in Membrane Structure Elucidation. Biochim. Biophys. Acta2009, 1788, 225-233. (95) Chmyrov, V.; Spielmann, T.; Hevekerl, H.; Widengren, J. Trans–Cis Isomerization of Lipophilic Dyes Probing Membrane Microviscosity in Biological Membranes and in Live Cells. Anal. Chem., 2015, 87, 5690–5697. (96) Sterling, S.M.; Allgeyer, E.S.; Fick, J.; Prudovsky, I.; Mason, M.D.; Neivandt, D.J. Phospholipid Diffusion Coefficients of Cushioned Model Membranes Determined via ZScan Fluorescence Correlation Spectroscopy. Langmuir, 2013, 29, 7966–7974. (97) Dey, S.; Mandal, U.; Mojumdar, S.S.; Mandal, A.K.; Bhattacharyya, K. Diffusion of Organic Dyes in Immobilized and Free Catanionic Vesicles. J. Phys. Chem. B, 2010, 114, 15506–15511. (98) Ghosh, S.; Adhikari, A.; Mojumdar, S.S.; Bhattacharyya, K. A Fluorescence Correlation Spectroscopy Study of the Diffusion of an Organic Dye in the Gel Phase and Fluid Phase of a Single Lipid Vesicle. J. Phys. Chem. B, 2010, 114, 5736–5741. (99) Menard-Moyon, C.; Venkatesh, V.; Krishna, K. V.; Bonachera,F.; Verma, S.; Bianco, A. Self-Assembly of Tyrosine into Controlled Supramolecular Nanostructures. Chem. - Eur. J.2015, 21, 11681−11686. (100) Perween, S.; Chandanshive, B.; Kotamarthi, H. C.; Khushalani, D. Single Amino Acid Based Self-Assembled Structure. Soft Matter2013, 9, 10141−10145. (101) Banik, D.; Kundu, S.; Banerjee, P.; Dutta, R.; Sarkar, N.Investigation of Fibril Forming Mechanisms of L‑Phenylalanine andL‑Tyrosine: Microscopic Insight toward Phenylketonuria and Tyrosinemia Type II. J. Phys. Chem. B2017, 121, 1533−1543. (102) Tiiman, A.; Jarvet, J.; Graslund, A.; Vukojevic ̈ , V. Heterogeneity ́ and Turnover of Intermediates during Amyloid-β (Aβ) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy. Biochemistry2015, 54, 7203−7211. (103) D’Amico, M.; Carlo, M. G. D.; Groenning, M.; Militello, V.; Vetri, V.; Leone, M.Thioflavin T Promotes Aβ(1–40) Amyloid Fibrils Formation. J. Phys. Chem. Lett., 2012, 3, 1596–1601. 40 ACS Paragon Plus Environment

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Biography

Niloy Kundu is currently pursuing his Ph.D degree in the Sarkar lab in the department of Chemistry at Indian Institute of Technology, Kharagpur. He received his B.Sc. degree from university of Calcutta in 2011 and M.Sc. degree in Chemistry from Indian Institute of Technology, Kharagpur, India in 2013. His current research focuses on ultrafast dynamics and single molecule spectroscopy in self assembled confined assemblies. Biography


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Debasis Banik is a research scholar in the Department of Chemistry at Indian Institute of Technology (IIT) Kharagpur. He received his B.Sc. degree in Chemistry from Hooghly Mohsin College under Burdwan University at 2011. In between 2011 to 2013, he did his M.Sc (Chemistry) from IIT Kharagpur and joined as PhD student under the Supervision of Professor Nilmoni Sarkar at 2013 June. His current research interest is single amino acid based selfassembly formation, ultrafast dynamics, excited-state intramolecular proton transfer processes. Biography

Nilmoni Sarkar is a Professor in the Department of Chemistry, Indian Institute of Technology (IIT), Kharagpur, India. He received his M.Sc. degree in Chemistry in 1986 from Burdwan University, Burdwan, India. From 1990 to 1994 he worked with Professor Kankan Bhattacharyya at the Indian Association for the Cultivation of Science (IACS) in Kolkata, India to receive his Ph.D. degree in 1994. He stayed in the same group as a Research Associate up to March, 1996. Subsequently, he spent March, 1996 to May, 1996 as a JSPS short-term postdoctoral fellow with Professor K. Yoshihara at Institute for Molecular Science (IMS), Japan. He worked with Professor H. Hayashi at The Institute of Physical and Chemical Research (RIKEN), Japan from June, 1996 to February, 1998. He worked with Professor T. Tahara at IMS from March, 1998 to June, 1998 before joining Chemistry Department IIT, Kharagpur in 1998. His research interest includes Fluorescence Spectroscopy and ultrafast chemical dynamics in organized assemblies. The current activities in his research group are focused on Time44 ACS Paragon Plus Environment

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Correlated Single Photon Counting (TCSPC), Femtosecond Fluorescence Up-Conversion, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM) techniques to study different photophysical and dynamical processes in organized molecular assemblies. He is also exploring the unique class of Surface Active Ionic Liquids (SAILs) and their applications to the formation of ionic liquid in oil microemulsions and vesicles spontaneously.

Professor Sarkar has supervised 16 Ph.D. students so far and published more than 185 research papers.


Self-Assembly of Amphiphiles into Vesicles and Fibrils - American

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