SELF-ASSEMBLED POLYSTYRENE-BLOCK-POLY (ETHYLENE OXIDE) (PS-b- PEO) MICELLE MORPHOLOGIES IN SOLUTION. A Dissertation. Presented to

SELF-ASSEMBLED POLYSTYRENE-BLOCK-POLY (ETHYLENE OXIDE) (PS-b- PEO) MICELLE MORPHOLOGIES IN SOLUTION A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Prachur Bhargava May 2007

SELF-ASSEMBLED POLYSTYRENE-BLOCK-POLY (ETHYLENE OXIDE) (PS-b- PEO) MICELLE MORPHOLOGIES IN SOLUTION Prachur Bhargava Dissertation Approved: Advisor Dr. Stephen Z. D. Cheng Accepted: Department Chair Dr. Mark D. Foster Committee Member Dr. Darrell H. Reneker Dean of the College Dr. George R. Newkome Committee Member Dr. W.L. Mattice Dean of the Graduate School Dr. George R. Newkome Committee Member Dr. Frank W. Harris Date Committee Member Dr. Alper Buldum ii

ABSTRACT In this research, we have investigated the self-assembly behavior of the amphiphilic diblock copolymer polystyrene-b-poly(ethylene oxide) PS 962 -b-peo 227 in DMF/water and DMF/acetonitrile mixtures. We have used solution conditions, namely copolymer concentration, solvent composition and temperature to control the morphology of aggregates. With increasing the water concentration in the DMF/water or the acetonitrile concentration in the DMF/acetonitrile system, the micelle morphology observed in transmission electron microscopy changed from spheres to worm-like cylinders and then, to vesicles. Critical micelle concentrations were determined by static light scattering. Morphological diagrams were constructed from the study of the micelle morphology changes in different copolymer concentrations. Based on the observations of morphological reversibility and annealing experiments, these two morphological diagrams were proven to be in thermodynamic equilibrium. Although the trend in morphological changes was identical in these two systems, there were remarkable differences in the morphological diagrams of PS 962 -b-peo 227. At higher copolymer concentrations a worm-network, which led to an order of magnitude increase in the inherent viscosity of the colloidal system was formed only in the DMF/water system. 4 The percentage of selective solvent required to induce morphological changes was much higher in case of DMF/acetonitrile system. The driving force for these morphological changes was understood to approach micelle free energy minimization. Approximate iii

micelle free energy calculations confirmed that the free energy decreases as the morphology changes from spheres to worm-like cylinders and then to vesicles with an increase in the selective solvent concentrations, and is dominated by the free energy of the interface. Further insight into the thermodynamic nature of morphological changes was achieved by inducing temperature driven morphological changes in DMF/water mixtures. With increasing the temperature, changes in the morphology from vesicles to worm-like cylinders and then to spheres were observed. Cooling the system back to room temperature regenerated the vesicle morphology indicating that the morphological changes are reversible. No hysteresis was observed in the morphological transitions during heating and cooling, indicating that the system is in thermodynamic equilibrium. The critical micellization temperatures and critical morphological transition temperatures were determined by turbidity measurements and were found to be dependent on the copolymer concentration and water content in the DMF/water mixture. The driving force for these morphological changes was understood to be reduction in the free energy of the corona and interfacial energy, which resulted in minimization of the micelle free energy. iv

DEDICATION This dissertation is dedicated to my grandparents, Dr. K.S. Bhargava and Mrs. Sheel Bhargava and my parents, Mr. Pradeep Bhargava and Dr. Keerti Bhargava. I am indebted to them for their support, guidance and encouragement. v

ACKNOWLEDGEMENTS I would like thank my advisor Dr. Stephen Z. D. Cheng for his invaluable guidance, believing in me and supporting me.i want to express my gratitude to my committee members Dr. Darrell H. Reneker, Dr. Frank W. Harris, Dr. Alper Buldum for their suggestions. I would like to thank my wife Dr. Sphurti Bhargava for her constant inspiration, helping with several experiments and useful discussions. I would like to thank my group members, especially Dr Kwang-Un-Jeong and Dr.Matthew J. Graham for their help on numerous occasions. Last, but not the least I want to thank all of my family back in India. vi

TABLE OF CONTENTS LIST OF TABLES..x Page LIST OF FIGURES....xi CHAPTER I. INTRODUCTION.... 1 II. BACKGROUND.5 2.1 Amphiphilic Molecules.......5 2.2 Amphiphilic block copolymers...7 2.3 Amphiphilic block copolymers vs small amphiphilic molecules...8 2.4 Applications of amphiphilic block copolymers and their self assembled micelle morphologies...9 2.5 Micelle structure and morphology 13 2.6 Methods to obtain micelles from amphiphilic block copolymers.16 2.6.1 Single Solvent Process 16 2.6.2 Two solvent or multi-solvent Solvent Process 17 2.7 Controlling Micelle Morphology & Size..18 2.7.1 Molecular parameters...19 2.7.2 Solution parameters. 21 vii

2.8 Background on micellization and studies of Polystyrene-b-Poly(ethylene oxide)..27 III. OBJECTIVES..32 IV.EXPERIMENTAL 33 4.1 Sample Synthesis...33 4.2 Preparation of micellar solutions 33 4.3 Transmission electron microscopy.35 4.4 Static Light Scattering Experiments...35 4.5 Turbidity measurements..36 4.6 Viscosity measurements..36 V. MORPHOLOGICAL CHANGES WITH SOLVENT COMPOSITION.....37 5.1 Molecular mechanism of morphological Changes...47 5.2 Thermodynamic and Kinetic Aspects of the Morphological Changes...49 VI. MICELLE FREE ENERGY CALCULATIONS...58 VII. EQUILBRIUM SIZES OF STRUCTURES..67 7.1 Form for F total....67 7.2 Exponents..68 7.3 Coefficients 69 VIII. PHASE MORPHOLOGY OF SELF-ASSEMBLED WORMS...72 IX. TEMPERATURE INDUCED MORPHOLOGICAL TRANSITIONS..81 9.1 Driving Force for the Morphological Changes..85 9.2 Initial Copolymer Concentration Effects on the Morphological Changes.89 9.3 Effect of Water Concentration on the Morphological Changes.....92 viii

9.4 Pathways of Morphological Changes in Isothermal Experiments...95 X. SUMMARY.....101 REFERENCES 104 ix

LIST OF TABLES Table Page 6.1 Micelle size and calculated parameters for a 0.5 (wt.) % initial copolymer concentration at three selective solvent concentrations...60 6.2 Calculated free energy components and total micelle free energies at three selective solvent concentrations...63 7.1 Calculated coefficients for the free energy dependence on size of structures at selective solvent concentrations given in... 69 9.1 Morphologies formed at different temperatures for different copolymer concentrations and 4.5 (wt.) % water concentration 91 x

LIST OF FIGURES e Figure Page 2.1 Structure of an amphiphilic molecule...5 2.2 Self assembled Amphiphilic molecule..6 2.3 Self- assembly of amphiphilic block copolymers 8 2.4 Block copolymer micelles for drug encapsulation...10 2.5 Drug loading in block copolymer micelles...11 2.6 Multiple morphologies formed by amphiphilic block copolymers..14 2.7 Formation of vesicles from amphiphilic block copolymers.....15 2.8 Multiple morphologies observed by transmission electron microscopy for Polystyrene-b-Poly(acrylic acid)..15 2.9 Multiple morphologies (a) Helical for PS 40 -b-piaa 10 (b) pearl necklace for PS(193k)-b-PDMS(39k) observed by transmission electron microscopy 16 2.10 Morphological Phase Diagram for polybutadiene-block-poly(ethylene oxide) (PBD-b-EO)...22 2.11 Morphological Phase Diagram for polystyrene-block-poly(acrylic acid) PS 310 -b-paa 52.. 23 2.12 Morphological Phase Diagram for of poly(acrylic acid)-(1,4)-polybutadiene diblock (PAA 75 -PBD 103 )..25 2.13 Effect of ion concentration on the morphology of PS-b-PEO.....28 2.14 Effect of ion PEO content on the morphology of PS-b-PEO......29 2.15 Tubules(a) and other complex bilayer aggregates (b)& (c) of PS 240 -b- PEO 15.. 30 xi

2.16 Vesicles with hollow rods for PS 100 -PEO 30... 30 2.17 Hexagonally packed hollow loops for PS-b-PEO 31 5.1 Morphological transitions with adding water in the DMF/water system for initial copolymer concentration of 0.1 (wt.) %. (a) a pure sphere morphology at 2.75 (wt.) % water; (b) a mixed spheres and rods at 3.14 (wt.) % water; (c) a mixed spheres and long rods at 3.54 (wt.) % water; (d) a pure worm-like morphology at 4.17 (wt.) % water; (e) a mixed worm-like and vesicles at 4.24 (wt.) % water; and (f) a pure vesicle morphology at 4.52 (wt.) % water...38 5.2 Morphological transitions with adding acetonitrile in the DMF/acetonitrile System for initial copolymer concentration of 0.5 (wt.) %. (a) a pure sphere morphology at 8 (wt.) % acetonitrile; (b) a mixed spheres and rods at 16 (wt.) % acetonitrile; (c) a mixed spheres and long rods at 30 (wt.) % acetonitrile; (d) a pure worm-like morphology at 37 (wt.) % acetonitrile; (e) a mixed worm-like and vesicles at 45 (wt.) % acetonitrile; and (f) a pure vesicle morphology at 70 (wt.) % acetonitrile.39 5.3 Static Light Scattering measurements with adding water in the DMF/water system. The arrows point out the cmc at different initial copolymer concentrations 41 5.4 Static Light Scattering measurements with adding acetonitrile in the DMF/ acetonitrile system. The arrows point out the cmc at different initial copolymer concentrations...42 5.5 Turbidity measurements with adding water in the DMF/water system. The arrows point out the cmc at different initial copolymer concentrations 43 5.6 Turbidity measurements with adding acetonitrile in the DMF/acetonitrile system. The arrows point out the cmc at different initial copolymer concentrations...44 5.7 Morphological change diagram for PS 962 -b-peo 227 in the DMF/water system....45 5.8 Morphological change diagram for PS962-b-PEO227 in the DMF/acetonitrile system 46 5.9 Representation of morphologies (a) spherical observed at low selective solvent concentration (b) interacting spheres observed at low selective solvent concentration.(c) short rod (d) long rod /worm like with spherical end caps (e) lamella.....47 xii

5.10 Reversible morphological changes by adding DMF into the DMF/acetonitrile system with 0.6 (wt.) % initial copolymer concentration and 68 (wt.) % acetonitrile. (a) the original micelle morphology is pure vesicle; (b) mixed worm-like and vesicles at 50 (wt.)% of acetonitrile; (c) a pure worm-like morphology at 37 (wt.) % acetonitrile; (d) mixed spheres and rods at 25 (wt.) % acetonitrile, and (e) a pure sphere morphology at 7 (wt.) % acetonitrile..51 5.11 Morphologies formed by a fast rate of acetonitrile addition to a 1 (wt.) % initial copolymer solution in DMF. Acetonitrile was added to 35 (wt.) % (a) after one day; (b) after two months...52 5.12 Morphologies formed by a fast rate of acetonitrile addition to a 2 (wt.) % initial copolymer solution in DMF. Acetonitrile was added to 68(wt.) % (a) after1 day; (b) after two months.53 5.13 Kinetically trapped morphologies formed by a fast rate of water addition to a 1 (wt.) % initial copolymer solution in DMF, water was added upto 3% wt.%.....54 5.14 Doughnut or toroid morphology formed by a fast rate of water addition to a 1 (wt.) % initial copolymer solution in DMF..55 5.15 Complex bilayer aggregates formed by continuous addition of water to DMF. 56 5.17 TEM images after shadowing with Platinum (a) spheres (b) a mixture of spheres and vesicles (c) & (d) worm like micelles..57 5.16 Porous spheres formed on adding 12wt% water to a 1.5wt% copolymer solution in THF..66 6.1 Free energy changes with acetonitrile content for various morphologies in the copolymer-dmf/acetonitrile system 66 7.1 Free energy changes with micelle size R for spheres in DMF/water system..70 7.2 Free energy changes with micelle size R for cylinders in DMF/water system 70 7.3 Free energy changes with micelle size R for lamellae in DMF/water system.70 8.1 Worm network formed in 4 (wt.) % copolymer concentration and 3.9 (wt.) % water content in DMF/water mixture(a) Y and T shaped junctions (b) entangled network (c) microscopic size (d) multiple junctions....73 8.2 Change in inherent viscosity on adding water in a 4 (wt.) % initial copolymer concentration solution in DMF....75 xiii

8.3 Aligned cylinders at a 2 (wt.) % initial copolymer concentration and a 34 (wt.)% acetonitrile in the DMF/acetonitrile system...76 8.4 Change in inherent viscosity on adding acetonitrile in a 4 (wt.) % initial copolymer concentration solution in DMF...77 8.5 Representation of the Y junction.....78 9.1 Morphological changes on heating a system with 0.2 (wt.) % copolymer and 4.5 (wt.)% water concentration in DMF/water: (a) pure vesicles formed at room temperature; (b) a mixture of vesicles and worm-like cylinders at 45 ºC; (c) pure worm-like cylinders at 50 ºC; (d) a mixture of spheres and rod-like cylinders at 60 ºC; (e) pure spheres at 70 ºC...82 9.2 Change in turbidity with temperature for a micelle system with 0.4 (wt.) % copolymer concentrations and 4.35 (wt.) % water concentration in DMF/water on heating and cooling.84 9.3 Intermediate morphologies formed on heating a system with 0.2 (wt.) % copolymer and 4.5 (wt.) % water concentration in DMF/water: (a) a mixture of vesicles and rings at 40 ºC; (b) lamellae with protruding rods at 45 ºC; (c) circular lamellae with protruding rods; (d) vesicles with protruding rod 85 9.4 Change in polymer solvent interaction parameter χp-s with temperature Individual polymer-solvent interaction parameters: χps-water, χps-dmf and χpeo-dmf...88 9.5 Change in polymer solvent interaction parameter χp-s with temperature polymer- DMF/water interaction parameters: χps-solvent and χpeo-solvent 89 9.6 Change in turbidity with temperature for micelle systems with 4.5 (wt.) % water concentration and different copolymer concentrations in DMF/water systems..90 9.7 Change in intensity of scattered light with temperature for micelle systems with 0.1 (wt.) % copolymer and three different water concentrations in DMF/water: 6 (wt.) %, 8 (wt.) % and 10 (wt.) %...93 9.8 Change in PS-solvent interaction parameter, χps-solvent, with temperature for different water concentrations in DMF/water systems 94 9.9 Rod-like cylindrical morphology formed from a system with 0.4 (wt.) % copolymer concentration and 4.35 (wt.) % water concentration in DMF/water in which the original morphology was vesicles: (a) after rapidly heating to 50 ºC and equilibrating for 2 hrs; (b) after rapidly heating to 80 ºC, equilibrating for 2 hrs and then, quenching to 50 ºC and equilibrating for another 2 hrs 96 xiv

9.10 After quenching a system with 0.4 (wt.) % copolymer concentration and 4.35 (wt.) % water concentration in DMF/water in which the original morphology was vesicles to 50 ºC, the morphological changes with time: (a) large compound micelles after 30 min; (b) mixed large compound micelles and rod-like cylindrical micelles after 50 min; (c) rod-like cylindrical micelles after 1h; and (d) rod-like cylindrical micelles after 2h...98 9.11 After rapidly heating a system with 0.4 (wt.) % copolymer concentration and 4.35 (wt.) % water concentration in DMF/water in which the original morphology was vesicles at room temperature to 65 ºC, equilibrating for 2h, then quenching to 50 ºC, the morphological changes with time: (a) large compound micelles after 30 min, (b) rod-like micelles after 1h, (c) rod-like micelles after 2h......99 9.12 After rapidly heating a system with 0.4 (wt.) % copolymer concentration and 4.35 (wt.) % water concentration in DMF/water in which the original morphology was vesicles at room temperature to 70 ºC, the morphological changes with time: (a) long rods and spheres after 15 min, (b) short rods and spheres after 30 min...100 xv

CHAPTER I INTRODUCTION Amphiphilic block copolymers have been known to self-assemble in solution to form a range of nanoscale morphologies 1, 2, 3, 4, 5, 6, 7, 8. These colloidal nanostructures have diverse applications such as carriers for drug delivery 9, 10, 11 and as nanotemplates 12. Key requirements for most applications include controlling the morphology and size of these self-assembled aggregates, which can be tuned via molecular 13 and solution parameters 14. Molecular parameters include the chemical repeat units, the relative block size, the molecular weight, and the polymer architecture. Solution parameters include solvent composition, polymer concentration, solution ph, temperature, and the presence of additives (ions, homopolymer, and surfactant). Most of the research work done in the last decade has focused on controlling the morphology of these aggregates by varying molecular parameters. Controlling the morphology by solution parameters offers the advantage of simplifying material synthesis. Only few research groups have used this approach and the method of preparation, and thermodynamic stability of these micelles has been a topic of discussion in the recent past 14,15. In this research, we have investigated the self-assembly behavior of an amphiphilic diblock copolymer, polystyrene-block-poly(ethylene oxide) (PS-b-PEO), in N,N-dimethylformamide (DMF)/water and DMF/acetonitrile 16. In both cases water and 1

acetonitrile are selective solvents for the PEO block. The degrees of polymerization of the PS and PEO blocks were 962 and 227 (PS 962 -b-peo 227 ), respectively. We have used solution conditions, namely copolymer concentration, solvent composition and temperature to control the morphology of aggregates. With increasing the water concentration in the DMF/water or the acetonitrile concentration in the DMF/acetonitrile system, the micelle morphology observed in transmission electron microscopy changed from spheres to worm-like cylinders and then, to vesicles. In between the concentration regions of two neighboring pure micelle morphologies, mixed morphologies such as spheres with short cylinders or worm-like cylinders with vesicles could be found. The morphological diagrams were constructed from the study of the micelle morphology changes in different copolymer concentrations and the critical micellization concentrations for both systems at different copolymer concentrations as determined by static light scattering experiments. Based on the observations of morphological reversibility and annealing experiments, these two morphological diagrams were proven to be in thermodynamic equilibrium. Although the trend in morphological changes was identical in these two systems, there were remarkable differences in the morphological diagrams of PS 962 -b-peo 227. At higher copolymer concentrations a worm-network, which led to an order of magnitude increase in the inherent viscosity of the colloidal system was formed only in the DMF/water system. The percentage of selective solvent required to induce morphological changes was much higher in case of DMF/acetonitrile system. These differences can be attributed to the large difference between the polymerselective solvent interaction parameters. The driving force for these morphological changes was understood to approach micelle free energy minimization. Approximate 2

micelle free energy calculations confirmed that the free energy decreases as the morphology changes from spheres to worm-like cylinders and then to vesicles with an increase in the selective solvent concentrations, and is dominated by the free energy of the interface. Further insight into the thermodynamic nature of morphological changes was achieved by inducing temperature driven morphological changes in DMF/water mixtures. With increasing the temperature, changes in the morphology from vesicles to worm-like cylinders and then to spheres were observed. Mixed morphologies like vesicles and rings, vesicles and worm-like cylinders and rod-like cylinders and spheres were also formed in the intermediate temperature regions. Cooling the system back to room temperature regenerated the vesicle morphology indicating that the morphological changes are reversible. No hysteresis was observed in the morphological transitions during heating and cooling, indicating that the system is in thermodynamic equilibrium. We also however, captured intermediate morphologies, which suggest the possible pathways for these changes. The critical micellization temperatures and critical morphological transition temperatures were determined by turbidity measurements and were found to be dependent on the copolymer concentration and water content in the DMF/water mixture. The morphological changes were only possible if the water content in the DMF/water mixture was low else the mobility of the polystyrene chains would be severely restricted. The driving force for these morphological changes was understood to be reduction in the interfacial energy and free energy of the corona, which resulted in minimization of the micelle free energy. Morphological observations at different time periods of isothermal experiments indicated that in the pathway of one equilibrium 3

morphology transfers to another, large compound micelles formed as an intermediate or metastable stage of this pathway. 4

CHAPTER II BACKGROUND 2.1 Amphiphilic Molecules Amphiphilic molecules are a special class of molecules, which have dual affinity or affinity for two or more different kind of environments. A simplest amphiphilic molecule is shown in Figure 1, which has a distinct hydrophilic head and a hydrophobic tail. Thus the dual affiliation is for hydrophilic and hydrophobic environments. Hydrophilic head group Hydrophobic hydrocarbon tail Figure 2.1 Structure of an amphiphilic molecule The most common example of amphiphilic molecules are surfactants which are present in the soaps and detergents which we use in everyday life. An example of a surfactant molecule is sodium dodecyl sulfate. Another class of amphiphilic molecules is lipids, 5

which are present in all living beings. An interesting property of these amphiphilic molecules is that they can self-assemble in solution to form a variety of structures. Selfassembly is defined as Spontaneous, reversible organization of pre-existing components by means of non-covalent interactions 17. These non-covalent interactions may include one or more of the following forces: hydrophobic forces, hydrogen bonding, van der Waals forces, and electrostatic forces. The self- assembly takes place at molecular concentrations above a critical concentration, which is known as the critical micelle concentration (CMC) Figure 2.2 shows some of the different self assembled structures small amphiphilic molecules can form. These include spheres, rods, monolayers, vesicles etc. Monolayer Vesicle Bilayer Sphere Figure 2.2 Self assembled Amphiphilic molecule In the human body, these molecules self-assemble to form a plethora of organized structures like the lipid bilayer membranes, which perform extremely sophisticated biological functions. These colloidal assemblies and their transformations are the very basis of many life processes. 6

2.2 Amphiphilic block copolymers This dual affiliation or amphiphilic nature can be incorporated into polymers by chemically linking together, blocks of two dissimilar repeating units to obtain amphiphilic block copolymers. Simplest case is when a hydrophobic block and a hydrophilic block are linked together. eg. polystyrene-block-poly(acrylic acid) PS-b-PAA where PS is hydrophobic and PAA is hydrophilic. Amphiphilic block copolymers can self-assemble to form micelles in aqueous media or when mixed with organic solvents. Micelles of block copolymers, which have a long corona and small core, are termed as star-like ; while, the ones with a large core and short corona are termed as crew cut. 18 Figure 2.3 shows a representation of a diblock copolymer and it s self-assembly. Graft block copolymers can also self assemble as shown in the figure. A plethora of morphologies have been recently reported, pioneered by Eisenberg and other research groups, some of which are analogous to those formed by small molecule amphiphiles, such as surfactants 13, 19, 20, 21, 22, 23, 24. Symmetric micelles, in which the core and corona forming blocks are similar in size, can also form multiple micelle morphologies. 24 We will review this in a later section. 2.3 Amphiphilic block copolymers vs small amphiphilic molecules Although small amphiphilic molecules and amphiphilic block copolymers share the same amphiphilic nature, there are several similarities and differences in their self-assembly behavior and properties of the self assembled aggregates. The similarity is that both these species can self-assemble under suitable conditions to form aggregates with multiple morphologies. The differences are as follows: 7

Figure 2.3 Self- assembly of amphiphilic block copolymers. 1. Size of molecules and the self assembled aggregates: Since polymer molecules are much larger than small amphiphiles, the size of block copolymer aggregates is usually larger as compared to the small amphiphiles. Also there is greater flexibility in tuning the size of these aggregates, which we will discuss in the later section. 2. Kinetic aspects: For small molecules the self-assembly process is instantaneous, while for amphiphilic block copolymers due to the large size kinetic aspects need to be considered and this adds another level of complexity to block copolymer self-assembly. 3. Degree of segregation of the core forming and corona block: In small molecules amphiphiles, the hydrocarbon tail and the hydrophobic head are strongly segregated. In case of amphiphilic block copolymer the degree of segregation can be tuned by choosing the chemical repeat units of the blocks. 8

4. Self-assembly polymorphism: Small molecule amphiphiles can form a variety of morphologies including spheres, rods, worm-like, bilayer vesicles etc. Biological amphiphiles like proteins can form complex helical morphologies. For block copolymers, work in many research groups has focused on obtaining multiple morphologies, which match the plethora of morphologies of small amphiphiles and biological amphiphiles. With the flexibility in designing block copolymers and their interesting self-assembly behavior in both aqueous and a variety of mixed solvents there is a potential of forming complex morphologies which may be not be possible for their small molecule counterparts. 5. Range of Applications: Self-assembled morphologies of amphiphilic block copolymers possess better stability and are more robust as compared with those formed by small molecule amphiphiles such as surfactants. This property coupled with the flexibility in designing them gives them potential for a range of applications, which are discussed, in the next section. The ratio of the hydrophobic part and hydrophilic can be easily tuned by choosing the molecular weight of the blocks. The flexibility of the aggregates can be tuned for a particular application by choosing the glass transition temperature of the blocks. Also the core forming block or the corona forming block can be selectively crosslinked to impart additional stability to these aggregates. 2.4 Applications of amphiphilic block copolymers and their self assembled micelle morphologies 1) Emulsifying agents and emulsion stabilizers: Amphiphilic block copolymers can be used as both emulsifying agents and emulsion stabilizers. For emulsifying agents, the 9

molecular weight is usually below 2000 while for post stabilization of emulsions higher molecular weight is required. Both steric effects and electrostatic effects can provide stabilization of emulsions. Block copolymers are being designed for emulsion polymerization such that the hydrophobic group matches with the latex polymers. This provides a maximum interaction between the hydrophobic block and the particle surface. Polymer (ethylene oxide)-block-poly(propylene oxide) copolymers have been investigated for the this application for different block lengths and ratios of the hydrophobic and hydrophilic block 25. Polystyrene-block-poly(ethylene oxide) (PS-b- PEO) has also been investigated as emulsion stabilizers 26. In related applications amphiphilic block copolymers can also be utilized as demulsifiers 27, defoamers 28 and foam stabilizers 29. 2) Drug delivery and Gene Delivery Self-assembled colloidal nanostructures of amphiphilic block copolymers have been investigated as vehicles for drug delivery for over a decade. Hydrophobic drugs can be incorporated in the hydrophobic core, while the hydrophilic shell provides steric stability Hydrophobic Drug Steric Stabilization Figure 2.4 Block copolymer micelles for drug encapsulation 10

and protects the drug from interacting with blood components 30 Poly(ethylene oxide) (PEO) based block copolymers with hydrophobic biodegradable polyesters (PLA, PGA etc.) and poly (amino acids) have been the highlight of last decade s research. Block copolymers with PEO in the corona are especially attractive due to its biocompatibility. 31 Small size of micelles in the range of 10-100nm make them attractive for this application. Enhanced permeation and retention effect displayed by these carriers can be used for passive delivery. Attaching ligands to the micelles can help to achieve active delivery and control the biodistribution. Drugs can be loaded in a variety of routes 32 as shown in Figure 2.5. Drugs have also been incorporated by covalently attaching them to the micelles. Drug release characteristics from micelles have also been studied and effort has been made in the literature to control the same and thus to obtain controlled release drug carriers. Figure 2.5 Drug loading in block copolymer micelles. 11

The cell uptake mechanism for the micelles is based on endocytosis 33. Recent developments include attaching ligands to micelles for receptor mediated endocytosis and developing ph sensitive micelles which can display the proton sponge effect. 34. Temperature driven drug delivery by thermo responsive micelles is also promising. This temperature dependent micellization and dimicellization is useful for temperature driven drug delivery. 35 Several block copolymers have been investigated in this respect including N-isopropylacrylamide based copolymers which is known to have a LCST behavior in water. 36, 37, 38 Block copolymers with a cationic block can be used to encapsulate DNA and serve as non-viral gene delivery vectors. Poly(ethylene imine) PEI and its copolymers, which display high cationic charge density and buffering capacity, have been researched extensively 39,40.The effect of morphology on the encapsulation efficiency and drug release has not been addressed in detail in literature. Most of the work done focuses on sphere morphology. Recently, worm like micelles and vesicles have also been investigated for encapsulating drugs and are anticipated to be an active research area in the future. 3) Applications in separation systems: The ability to form micelles by amphiphilic block copolymers can be used to solubilize hydrophobic compounds in the interior of the micelles. These molecules are an alternative to organic solvents for removal of organic molecules. Water based solutions of copolymers are also environmentally friendly. Temperature or ph induced association can be used as an external influence to trigger the separation process by inducing self- association or micellization by changing the stimuli. These stimuli responsive systems have potential use in the field of biotechnology in 12

which they can be used for extraction of sensitive bio-molecules like proteins. By external controls these species can be triggered to capture and release bio-molecules. The flexibility in design and possibility of attaching target ligands make them more attractive for these applications. PEO-PPO-PEO block copolymers have been utilized in these applications and investigated for structure property relationships. 41,42 Self- assembled micelles can also be used for separation of microorganisms from fluids and wastes 43 where microorganisms can be immobilized on the micelle surface. 4) Other Applications: Amphiphilic block copolymers can also be utilized in biosensors by immobilizing enzymes etc on their surface where their high specific surface area can be utilized. The self-assembly can also be used to obtain nanotemplates 12 and utilized for pattering etc. 2.5 Micelle structure and morphology Similar to small amphiphilic molecules amphiphilic block copolymers can also form a variety of morphologies. Figure 2.6 shows some of the different morphologies which can be obtained 44. In this schematic the grey part corresponds to the hydrophobic core and the individual black lines correspond to the hydrophilic chains. The morphologies in this figure are spherical, rod-like, interconnected rods, lamella, vesicles and large compound micelles (LCM). Eisenberg and co-workers investigated the internal structure of the LCMs. The bulk of the micelle consists of reverse micelles, which have the hydrophobic chains in the corona and hydrophilic chains in the core. The surface of these LCMs is stabilized by individual chains with hydrophilic chains on the surface. Let us look in detail at the vesicular morphology. As shown in Figure 2.7, one series of amphiphilic block copolymers can self-assemble to form one layer and another series of molecules 13

can form another layer and thus they can from bilayers which can be in the form of huge sheets and lamellae These lamella can close to form hollow structures; vesicles. The closure of the lamella is governed by the interaction of rim energy and bending energy 45. This will be discussed in detail in the section VI. Transmission electron microscopy (TEM) is a powerful tool to visualize directly these self-assembled morphologies. Figure 2.8 shows the multiple morphologies observed by transmission electron microscopy for polystyrene-b-poly(acrylic acid). In figures 2.8a-d spheres, rod, vesicles and large compound micelles can be observed respectively. 46 Other morphologies have also been reported. Sphere Cylinder/Rod Bicontinuous Rods Lamella Hydrophilic block hydrophilic in corona hydrophobic Vesicle Hydrophobic block Core of reverse micelles (hydrophilic) Large Compound Micelles Figure 2.6 Multiple morphologies formed by amphiphilic block copolymers. 14

Figure 2.7 Formation of vesicles from amphiphilic block copolymers. 100 nm 100 nm. 100 nm 1 µm Figure 2.8 Multiple morphologies observed by transmission electron microscopy for Polystyrene-b-Poly(acrylic acid). Helical morphology has been observed for a polystyrene-b-poly(isocyanopeptide) 47 as shown in Figure2.9a and pearl necklace morphology (Figure 2.9b) for Polystrene-bpolymer(dimethylsiloxane) PS(193k)-b-PDMS(39k) 48 15

2.6 Methods to obtain micelles from amphiphilic block copolymers Depending on the physiochemical nature of the block copolymer, micelles can be obtained by various routes. Below we discuss the various methods and give specific examples where the method has been used. A B 2000 Figure 2.9 Multiple morphologies (a) Helical for PS 40 -b-piaa 10 (b) pearl necklace for PS(193k)-b-PDMS(39k) observed by transmission electron microscopy. 2.6.1 Single Solvent Process In this method the block copolymer is dispersed in a selective solvent for one of the blocks and micelles are formed with the phobic block in the core and the philic block in the corona. This method is usually used for block copolymers in which the core forming block is flexible or its glass transition temperature is below the micellization temperature. This process can also be used if the core-forming block is glassy at the micellization temperature but is small as compared to the corona-forming block i.e for 16

star-like micelles with glassy cores. For example, Bates and co-workers 13 obtain micellar solutions of PBD-b-PEO by directly dispersing the block copolymer in water at room temperature which is a selective solvent for PEO. PBD is hydrophobic but is flexible at room temperature, and thus this process can be used. In this way, the polymer-solvent interaction parameter, χ P-S, does not vary. Another variation of this method is to dissolve both the blocks by heating a selective solvent and the cooling the system to induce micellization. Eisenberg and coworkers used this method to prepare micelles for polystyrene-b-poly(acrylic acid) in low alkanols (i.e. methanol to n-butanol) 49 2.6.2 Two solvent or multi-solvent process In block copolymers where the core-forming block is glassy at the micellization temperature, it is not possible to obtain stable micelle morphologies by the single solvent process. Either the block copolymer will not disperse or one would obtain kinetically trapped morphologies if an attempt were made to use the single solvent process for such block copolymers. In these cases a two or multi-solvent process is used. In the first step, the block copolymer is dissolved in a good solvent for both the blocks. Then a solvent is added which is selective for one of the blocks, which is miscible with the common solvent. As the selective solvent is added the solvent composition continuously changes and thus the he χ P-S in this method changes. At a critical concentration of the selective solvent, micellization is induced and micelles are formed. This critical solvent concentration can be determined using static light scattering and turbidity measurements. One example is polystyrene-block-poly(acrylic acid) (PS 310 -b-paa 52 ) dissolved in dioxane as a common solvent, and a then selective solvent for PAA. Water is added 17

slowly to induce micellization 14,50. Other block polymers such as PS-b-PDMS and PS-b- PEO have also been studied for self- assembly using this method Both the above methods offer material and process parameters which can be used to control the size and morphology of the self-assembled aggregates which will be discussed in the next section. 2.7 Controlling Micelle Morphology & Size The size and morphology of these self-assembled aggregates can be controlled by molecular parameters, which are pertaining to the chemistry of the block copolymer, and solution parameters, which are related to the characteristics of the solution. The molecular parameters include the nature of chemical repeating units of the blocks, the sizes of each block, the overall molecular weight, and the architecture. It has been shown in the past decade that these parameters are key to tune the micelle morphologies. 12,22. In most cases when these parameters are varied to control the morphology and size, the single solvent process is used. The second category of the parameters, which have been investigated only recently, are the type of solvents and solvent quality, the solvent/nonsolvent ratio, the copolymer concentration, the ph value, additives such as salts, ions and homopolymer and the temperature. 48,50. When solvent quality and solvent/non-solvent ratio are used as controlling parameters, mostly two solvent method is used. The micelle morphology is also sometimes tuned by introducing specific interactions like hydrogen bonding or excess chirality in the monomer units. 47 Below we discuss the above parameters in detail and cite some examples from literature. The formation of different morphologies is explained by considering the micelle free energy, which consists of three components. 46 The free energy of the core, which relates 18

to the stretching of the core-forming block; the free energy of the interface, which relates to the surface tension between the core forming block and the solvent; and the free energy of the corona, to which the electrostatic and steric interactions of the coronaforming blocks contribute. Mathematically this can be expressed as F = F core + F interface + F corona (2.1) Many researchers have also theoretically treated the micelle free energy 51, 52, 53, 54. Below we discuss the above parameters in detail and cite some examples from literature and discuss how some of these parameters affect morphology of the block copolymer aggregates. 2.7.1 Controlling Micelle Morphology & Size: Molecular parameters (a)chemical repeat unit of the two blocks: The chemical nature of the repeat unit determines interaction parameter χ between the two blocks. This would determine the degree of segregation of the two blocks. Usually hydrophobic and hydrophilic blocks linked together are super segregated 45. The chemical repeat units would also decide specific interactions like hydrogen bonding. Ionic or protonable groups present in the backbone and/or side chains would result in additional electrostatic interactions which could affect the free energy of the corona. Many different chemical repeat units and combinations have been investigated, some examples will be discussed in the sections below. (b) Copolymer composition: This relates to the ratio of the hydrophobic block and the hydrophilic block. This one of the most important factor to control the morphology of these self-assembled aggregates. Eisenberg and coworkers studied the effect of poly(acrylic acid) content in polystyrene-block-poly(acrylic acid) on the morphology of 19

the block copolymer 22. They used a two solvent method.in the first step the block copolymer was dissolved in N,N-dimethylformamide (DMF) which is a common solvent for both the blocks, then water was added drop wise until 25wt% water was reached. The resulting colloidal solution were placed in dialysis bags and dialyzed against water to remove the DMF. The initial copolymer concentration in DMF was kept fixed to 2wt%. They found that as the PAA content in the block copolymer decreased, the morphology of the aggregates changed from spheres to cylinders, to bilayer (lamella and vesicles) and eventually to compound micelles. The aggregates were analyzed by transmission electron microscopy. The multiple morphologies are shown in Figure 2.8. A systematic study, on the effect of block copolymer composition and molecular weight on the morphology of polybutadiene-block-poly(ethylene oxide) (PBD-b-PEO) has been carried out by Bates and coworkers 13. They varied the PEO content in the block copolymer and the molecular weight of the PBD block. The micelles were obtained by dispersing the block copolymer in water at room temperature, which is a selective solvent for the PEO block. This method could be used, as PBD is flexible at room temperature. They obtained a morphological phase diagram, which maps equilibrium micelle morphologies as a function of the varying parameters. The morphologies obtained and the morphological phase diagram is shown in Figure 2.10. They found that on decreasing the PEO content the morphology changed from spheres to worm like micelles and then to bilayers. They observed that the worm like micelles formed a network for a degree of polymerization of the PBD of 170 and a weight fraction of PEO of 0.34. 20

2.7.2 Controlling Micelle Morphology & Size: Solution parameters (a) Solvent and concentration: The effect of solvent is usually studied by using two solvent approaches. The effect of solvent can be divided into three parts. First, is the effect of solvent/non solvent ratio or the solvent composition, second the nature of the common solvent, and third the effect of selective solvent. Eisenberg and workers have studied the effect of solvent composition and common solvent on the block copolymer morphologies of PS-b-PAA 14,50, however the effect of different selective solvents has not been studied so far on any block copolymer system. The effect of solvent composition has been studied in terms of different water contents in dioxane/ water mixture. A two solvent process was used and the morphology was visualized by TEM. They found that on increasing the water content in the dioxane/water mixture the morphology changed from spheres to a mixture of spheres and cylinders, to pure cylinders, then to a mixture of cylinders and vesicles and finally to pure vesicles. They also studied the effect of copolymer concentration and obtained a morphological phase diagram which is shown in Fig 2.11. Varying the solvent composition changes the polymer solvent interaction parameter χ which has and effect on the interfacial free energy and thus affects the morphology. This is the only polymer and solvent system for which a study had been carried out. Very recently, Bang and workers studied the morphology for polystyrene-bpoly(isoprene) PS(13000)-b-PI(71000)in different solvent mixtures of di-n-butyl phthalate(dbp), diethyl phthalate (DEP), and dimethyl phthalate (DMP) 55. They obtained sphere, rods and vesicle morphology by varying the solvent composition. 21

N PB w PEO Figure 2.10 Morphological Phase Diagram for polybutadiene-block-poly(ethylene oxide) (PBD-b-PEO) However they obtained mostly mixed morphology in the compositions they investigated. The morphology was studied by SAXS( small angle X-ray scattering ) and cryo TEM. Eisenberg and coworkers studied the effect of common solvent on the morphology of PS-b-PAA. 46 The morphology depends also on the nature of the common solvent in which the copolymers are initially dissolved For 2% PS(500)-b-PAA(60) copolymers, when pure DMF is used as the common solvent, spherical micelles were formed; however, if the initial common solvent is THF or dioxane, vesicles are produced. 22

Spheres + Rods Spheres Rods Rods+Vesicles Vesicles Copolymer Concentration (wt%) Water content (wt%) Figure 2.11 Morphological Phase Diagram for polystyrene-block-poly(acrylic acid) PS 310 -b-paa 52. The choice of common solvent determines the dielectric constant of the medium and would effect the inter-corona interactions, in addition to affecting the polymer solvent interaction parameter χ. This would also effect the swelling of the core and thus the free energy of the core. Putuax and coworkers studied the effect of the copolymer concentration and architecture on the morphology of micelles of poly(styrene-b-isoprene) 23.For linear chains spherical 23

micelles were obtained, while for similar cyclic copolymer chains giant worm like micelles and vesicles were obtained. On increasing the copolymer concentration the morphology changed from planar particles to worm like micelles and then to vesicles at higher concentrations. (b) Effect of additives: Additives, which can be added, include salts such as sodium chloride (NaCl), calcium chloride (CaCl 2 ), surfactants such as sodium dodecyl sulfate and homopolymer. Also alkali such as sodium hydroxide (NaOH) and acid such as HCl can also be added to tune the morphology. Eisenberg and coworkers studied the morphogenic effect of the addition of salts such as CaCl 2 and NaCl on the micelles 46. As the concentration of added salt increases, the morphologies change from spheres to vesicles, which is similar to the effect of adding HCl, increasing the copolymer concentration or decreasing the length of soluble block. However, changes in the aggregate morphology can be achieved with much lower concentrations of CaCl 2 than of NaCl. An example is the morphology of the aggregates made from 1 wt% PS(410)-b- PAA(25)copolymers in DMF. Without any additives, the aggregates are small spheres. When NaCl is added to a final concentration of 10mM, vesicles are formed. However, if CaCl 2 is used as the additive, vesicles are formed when the added final concentration of CaCl 2 is as low as 90 mm. Discher and coworkers studied the effect of ph, NaCl and CaCl 2 concentration on the morphology of poly(acrylic acid)-(1,4)-polybutadiene diblock (PAA 75 -PBD 103 ) in detail 24. They obtained a morphological phase diagram which is shown in Figure 2.12. They observed spheres, worm like, a worm network above an additive concentration and higher order vesicles. Decreasing the ph or increasing the salt 24

content the morphology changed from spheres, to a mixture of spheres and cylinders, then to cylindrical network and finally to vesicles. Figure 2.12 Morphological Phase Diagram for of poly(acrylic acid)-(1,4)-polybutadiene diblock (PAA 75 -PBD 103 )Eisenberg and coworkers studied the effect of homopolymer styrene content in the PS-b-PAA 46. For a block copolymer which formed vesicles, adding homopolymer styrene changed the morphology from vesicles to rods and then to spheres.c) Effect of temperature Temperature has been used as a tool to control the block copolymer self-assembly for several years. 56, 57, 58, 59, 60, 61, 62, 63 Depending on the nature of the polymer-solvent interactions, if the system displays an upper critical solution temperature (UCST) behavior, micellization takes place below a critical temperature; while if the polymer displays a lower critical solution temperature (LCST) behavior micellization takes place 25

above a critical temperature. In either case this temperature is known as the critical micellization temperature (CMT). This temperature dependent micellization and dimicellization is specifically useful for temperature driven drug delivery. As mentioned earlier, N-isopropylacrylamide based copolymers have been investigated in this respect which is known to have a LCST behavior in water. 36,37,38 Poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-b- PPO-b-PEO) triblock copolymer has also been exhaustively investigated due its interesting LCST behavior in water. 64, 65, 66, 67, 68, 69 At higher concentrations it also exhibits ordered phases such as cubic, hexagonal and lamellar phases. 65 Relatively less literature is available on using temperature to control micelle morphologies of block copolymers in dilute solution. 48,58 Pispas and Hadjichristidis investigated the effect of temperature on polybutadiene-block-poly(ethylene oxide) (PBD-b-PEO) micelle solutions in water by static and dynamic light scattering (SLS and DLS). 70 They observed a slight increase in the intensity of scattered light and a decrease in the hydrodynamic radius with increasing temperature. But they could not conclude any changes in the micelle morphology. A temperature-concentration phase diagram was mapped for polystyrene-blockpolydimethlysilocsine) (PS-b-PDMS) in which PS formed the core and PDMS formed the corona by Iyama et al. 58 Using SLS and DLS they estimated distinct phases of spheres and cylinders on changing the temperature and polymer concentration. The effect of temperature on micelle morphology has so far mostly been studied by indirect techniques such as SANS, SLS and DLS. Very recently, temperature-induced morphological changes were studied by atomic force microscopy (AFM) for polystyrene-blockpolyisoprene (PS-b-PI) in a selective solvent for PI blocks. 71 The copolymer composition 26