Dipartimento di Chimica e Chimica Industriale
Corso di Laurea Magistrale in Chimica Industriale
Tesi di Laurea Magistrale
O
LIGO
(
ETHYLENE GLYCOL
)
MODIFIED
POLY
(
PENTAFLUOROSTYRENE
):
SYNTHESIS AND
CHARACTERIZATION OF THE SELF
-
ASSEMBLY
BEHAVIOR IN SOLUTION
RELATORI CONTRORELATORE
Prof. Giancarlo GALLI
Prof.ssa Celia DUCE
Dott.ssa Elisa MARTINELLI
CANDIDATO
Matteo CALOSI
Amphiphilic tetrafluorostyrene monomers (EFSn) carrying in para position an oligoethylene glycol chain with different lengths (n = 3-13) were synthesized and used for the preparation of the corresponding amphiphilic homopolymers (pEFSn-x) having different degrees of polymerization (x = 8-135) by a controlled radical polymerization reaction, such as ARGET-ATRP. The self-assembly behavior of monomers and homopolymers in water and organic solvents was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). In particular, DLS measurements on water solutions of the water-soluble homopolymers at room temperature evidenced the presence of nanoassemblies with hydrodynamic radius Rh = 2.5 − 6 nm, compatible with
the formation of unimer micelles. These nanoassemblies were proven to be disrupted by the addition of THF to the water/polymer solution. SAXS measurements revealed that self-folded nanoassemblies were compact globular spheres, with radius of gyration Rg in
the range 1.7 − 2 nm, corresponding to radii of a spherical particle in the range 2 − 3 nm. The Rg/Rh radius was <0.5, consistent with a core-shell nanostructure. On heating
above a critical temperature, identified as a cloud point temperature by light transmittance measurements, multi-chain microassemblies were formed, which reverted to nanoassemblies on cooling below this critical temperature. This LCST-type thermoresponsive transition was fully and sharply reversible.
Differential scanning calorimetry (DSC) analysis revealed that the homopolymers with n 8 were amorphous, while those with n = 13 were semicrystalline, owing to the crystallization of the longer oxyethylenic side chains. In any case the glass transition temperatures (Tg) were lower than room temperature, indicating that the EFSn backbone
featured a relatively high flexibility. Moreover, thermogravimetric analysis (TGA) showed that the polymer backbone possessed an increased thermal stability with respect to the corresponding oligoethylene glycol side chain.
Wettability and surface composition of the homopolymers were investigated by static contact angle and X-ray photoelectron spectroscopy (XPS) measurements. XPS measurements confirmed the presence of fluorine at the film surface, even though its concentration was lower than the theoretical amount, thus indicating a low effectiveness in surface migration of the fluorinated backbone. Consistently, homopolymer films exhibited moderate to low hydrophobicity as estimated by the measurements of water and n-hexadecane contact angles. However, the initial water contact angle and the film ability to respond to the wetting liquid were generally correlated to the hydrophilic/hydrophobic balance of the specific repeating unit of the polymer.
1 Introduction ... 1
1.1 Single-chain polymer nanoparticles ... 1
1.2 Self-folding amphiphilic polymers ... 2
1.3 Thermoresponsiveness of amphiphilic polymers ... 7
1.4 Characterization of single-chain polymer nanoparticles ... 8
1.5 Applications of single-chain polymer nanoparticles ... 9
1.6 Reversible-deactivation radical polymerization... 11
1.7 Properties of fluorinated polymers ... 16
1.8 Properties of oxyethylenic polymers ... 18
2 Objectives of the work ... 20
3 Results and discussion ... 23
3.1 Monomer synthesis ... 23
3.2 Polymer synthesis ... 26
3.3 Solubility properties of amphiphilic monomers and polymers... 31
3.4 Thermal properties of amphiphilic monomers and polymers in solution ... 33
3.5 Single-chain folding and self-assembly of amphiphilic polymers in solution ... 38
3.6 Thermal properties of amphiphilic polymers and monomers in bulk ... 52
3.7 Surface properties of amphiphilic polymer thin films ... 59
4 Concluding remarks ... 65
5 Experimental ... 68
5.1 Materials ... 68
5.2 Synthesis ... 70
5.3 Preparation of thin polymer films ... 81
5.4 Characterization ... 81
Introduction
1
1 Introduction
1.1 Single-chain polymer nanoparticles
Many natural macromolecules, e.g. enzymes, function as discrete objects with highly specific properties and functions deriving from a complex and well-defined chain structure capable under certain stimuli of self-assembling in the form required by such functions. As a contrast, synthetic polymer materials generally derive their properties from the collective behavior of a great number of macromolecular chains forming a myriad of intermolecular interactions that lead to the desired micro- and macroscopic properties.
The synthesis of highly defined and functional three-dimensional structures with a monodisperse molecular weight distribution, such as natural polypeptide chains, remains beyond the reach of synthetic macromolecular chemistry. Nevertheless, a series of alternative approaches has led to the synthesis of polymers capable of self-assembling and forming by such processes compact nano-sized functional three-dimensional structures. Such single chain polymer nanoparticles (SNCPs) can be obtained by reducing the conformational freedom of the macromolecular chain via various kinds of intramolecular interactions, both covalent and non-covalent1,2.
The origin of this kind of structure can be traced to dendrimers and other highly branched polymer chain structures developed since the 1980s. SCNPs have since then also been widely obtained from linear polymer chains possessing the necessary intramolecular bond formation capability3. The self-folding of such
chains through the formation of covalent intramolecular bonds has been the first option to be explored, leading to the collapse of the chain into a defined structure. A number of possible intramolecular covalent non-reversible cross-linking reactions have been developed4 for the purpose. Such macromolecules fall outside the scope of this thesis.
2
Another approach is the self-assembly of the single polymeric chain through reversible folding. Such macromolecules have a stimuli-responsive and tunable character that more closely approaches the general characteristics of functional single-chain polymeric systems found in natural systems. The first report of this kind of SCNP was in 2008 and used benzamide dimerization through hydrogen bonding to create reversible intramolecular self-assembly5. Hydrogen bonds are one of the key bases of self-assembly in natural systems and have been widely studied in the synthesis of SCNPs, being are regarded as the most prominent non-covalent binding class2. Other types of reversible crosslinking possibilities that have been explored, often also taking inspiration from interactions featured in natural systems, including π–π interactions6, host-guest systems7, metal-ligand interactions8 and hydrophobic interactions, notably for amphiphilic polymers in water solutions. The latter example will be explored further.
1.2 Self-folding amphiphilic polymers
A limitation of the classic design of SCNPs is that the polymer chains so obtained are generally not soluble in water and are capable of reversible self-folding only in organic solvents, unlike many of the natural macromolecules which inspired the concept. Conventional SCNPs can be made water-soluble by post-formation modification but this operation is non-reversible. Direct crosslinking of water-soluble polymers in aqueous environment, both reversible and non-reversible, has also been developed to create SCNPs and has proved effective, though limited by the requirement that the cross-linking reaction be possible in water (Figure 1-1)9.
A method developed throughout the last decade to assemble water-soluble SCNPs is through solvent-induced self-folding. In this kind of particles, no further chemical reactions are carried out after polymer synthesis, but the folding of the chains into particles happens after dissolving the polymer in water through hydrophobic attractive interactions between parts of a polymeric chain having such properties, while a hydrophilic part provides water solubility (Figure 1-2).
Introduction
3
Figure 1-1 Routes towards water-soluble SCNPs, reproduced from ref9.
The possible advantages of this technique are both practical, in that SCNPs can be obtained directly at high concentrations by simply dissolving a polymer precursor in water, and theoretical, in that the type of hydrophobic interactions exploited to lead to self-folding are of the same kind many natural macromolecules use to carry out their own self-assembly.
This kind of amphiphilic polymers is commonly obtained via copolymerization of hydrophobic and hydrophilic monomers. The distribution and ratio of the monomeric units determines the self-assembly process of the copolymers in an aqueous medium. It has been observed that block copolymers, alternating hydrophobic and hydrophilic chains, tend to form multi-macromolecular aggregate micelles or vesicles, with similar blocks in distinct polymeric chains forming intermolecular interactions10. Block copolymers have generally been prevalent in the study of self-assembly in water due to the existence of direct structure-property correlations providing guidelines to the assembly behavior, depending on the nature and size of the blocks involved. The ultimate properties of random amphiphilic copolymers, where hydrophobic and hydrophilic properties are statistically distributed throughout the chain, are harder to predict. However, random amphiphilic copolymers tend to assemble via intramolecular rather than intermolecular interactions, leading to the possibility of obtaining self-assembly by discrete macromolecular chains in water in globular structures generally termed unimer micelles11.
4
The first studied systems which generate unimer micelles in water have been polyelectrolytes12. In subsequent developments the most commonly used hydrophilic elements used for self-folding purposes have been oxyethilenic chains introduced into monomers capable of free radical chain polymerization. The key monomer in this field has been poly (ethylene glycol) methacrylate (PEGMA). First introduced by Terashima and colleagues in 201113,
PEGMA-based neutral copolymers have the advantage, compared to polyelectrolytes, of being soluble in a range of organic solvents in addition to water while also exhibiting self-folding capabilities in some of such solvents. This first copolymer featured a methacrylate which included a benzene-1,3,5-tricarboxamide (BTA) functional group at the end of an aliphatic side chain as a hydrophobic comonomer. Self-folding was achieved because of both hydrophobic interactions and hydrogen bonding between the BTA units (Figure 1-3)13. Further study would later show that BTA hydrogen bonding interactions were not necessary for self-folding but hydrophobic interactions were sufficient14. The effective existence of
Figure 1-2 Schematic representation of unimer micelle formation by self-assembly in water.
Introduction
5
folded unimer micelles rather than a random coil structure was proved in this first study by the observation of dots with 3-4 nm diameter by cryo-TEM.
Figure 1-3 Structure and self-assembly of PEGMA/BTAMA random copolymers, adapted from ref13.
Later developments have focused on using methacrylates with aliphatic or perfluorinated side chains of varying length as hydrophobic comonomers (Figure 1-4), generally showing nanostructures with hydrodynamic diameters lower than to 10 nm15,16. Random copolymers of PEGMA and 2-(perfluorohexyl)ethyl methacrylate have also been studied by small angle neutron scattering (SANS) and found to fold into prolate spheroid particles17. The same system, with the addition of a fluorescent rotor comonomer capable of giving different responses when confined within a micelle rather than in solution, was studied via fluorescence emission spectroscopy proving the effective micellar structure of the particles by inclusion of the rotor within the structure18. Lately methacrylates carrying poly(siloxane) as a side chain have also been used as hydrophobic comonomers to form unimer micelles19. Analogous amphiphilic polymers have been synthesized using an acrylamide rather than a methacrylate polymeric backbone, with PEG and alkyl pendants providing amphiphilicity20. A different approach, where an amphiphilic copolymer was obtained by grafting poly(vinyl acetate) on PEG chains, has also been shown to result in single-chain globular particles21.
6
Figure 1-4 Examples of amphiphilic random copolymers using methacrylates with alkyl or perfluorinated side chains as hydrophobic comonomers, adapted from refs15,16.
The globular conformation that seems to be often adopted by amphiphilic random copolymers in water stands in contrast to that of many other types of chain collapse by intramolecular crosslinking in organic solvents, which has been shown to lead in most cases to a sparse structure, where small, locally globular regions of the polymer chain are linked by random-coil sections in a structure resembling that of intrinsically disordered rather than globular proteins (Figure 1-5)22.
Figure 1-5 General structure of sparse versus globular SCNPs, reproduced from ref23
The polymers described also generally exhibit thermo-responsive solubility properties in water or other solvents. PEG-based polymers often show lower
Introduction
7
critical solution temperature (LCST) phase separation in water caused by the aggregation of polymer chains into macro-aggregates. Both aliphatic and fluorinated side chains in comonomers lead to such a situation, with the phase transition at the cloud point (Cp) depending on comonomer ratio and composition16,24.
When aliphatic pendants were used to confer hydrophobic character, a distinction was noted between two types of self-assembly in water: unimer particles which vary size depending on degree of polymerization, and multi-chain aggregates which possessed a fixed size. Comonomer ratio and aliphatic chain length determined the type of self-assembly25,26.
An alternative approach to create amphiphilic polymers was followed in this thesis work. It consists in including both the hydrophobic and hydrophilic functional groups within one structure of an amphiphilic monomer, which was then polymerized leading to an amphiphilic homopolymer.
1.3 Thermoresponsiveness of amphiphilic polymers
Thermo-responsive polymers are a category of stimuli-responsive macromolecules that can react to changes in temperature with a modification of their properties. In the field of amphiphilic polymers, thermoresponsiveness is often expressed through a drastic change in their solubility and aggregation behavior in water or other solvents. Most of such thermo-responsive polymers display a lower critical solution temperature (LCST) in aqueous solution. At temperatures below the LCST, polymers are soluble in water and exist as single discrete chains that in the case of “ordinary” amphiphilic polymers assume a random coil conformation due to hydrogen bonds between water and the hydrophilic segments of the polymer chain being the dominant form of interaction. When temperature is higher than the LCST, these interactions are disrupted and interactions between the hydrophobic segments become dominant27. This causes polymer chains to collapse and form aggregates, which are insoluble and form a cloudy dispersion (Figure 1-6). The balance between segment-water and
8
segment-segment interactions determines LCST. Structural factors that increase segment interactions decrease LCST, while those increasing segment-water interactions increase LCST28.
Poly(N-isopropylacrylamide) (PNIPAAm) and PEGMA are examples of polymers with a well-known LCST in water28. The behavior observed in amphiphilic random copolymers in water differs from this in the fact that the structure assumed below LCST is still globular rather than random coil and may be composed of a single polymer chain or a small number of chains. Cloud point transition still results in macroaggregates involving a large number of macromolecules.
Figure 1-6 Common behavior of thermoresponsive polymers, in this case shown for PNIPAAm, reproduced from ref29
1.4 Characterization of single-chain polymer nanoparticles
The main techniques used for characterization of nanoparticles are used for SCNPs, though their small size poses some problems.
Most commonly size measurements for SCNPs in solution are obtained by dynamic light scattering (DLS), which determines their hydrodynamic radius (Rh) based on the fluctuation of the scattering intensity created by their diffusion. The small size of the analyzed particles can be a problem for this technique as intensity of the scattered light is dependent on particle diameter to the 6th power.
Introduction
9
To circumvent the influence of larger aggregates, which may be only a very small percentage of the particle population, on measurements, DLS for SCNPs often uses volume or number plots under the assumptions of Mie theory, which avoids the problem with larger particles but makes the distribution more error prone. Diffusion ordered spectroscopy (DOSY) NMR analyzes size of particles based on their diffusion without depending on scattering and can supplement DLS data. DLS also does not provide direct information on the shape taken by nanoparticles. Small-angle neutron scattering (SANS) and small-angle x-ray scattering (SAXS) can provide the gyration radius (Rg) of the particles, as long as this is below 10 nm, and fitting of the form factor can attribute a geometrical shape (spherical, prolate, rod-like). Detailed imaging of SCNPs can be obtained by techniques such as atomic force microscopy (AFM) and transmission electron microscopy (TEM), but these methods are at the limits of their resolution and offer information only on dry state particles which prevents the study of solvent-induced folding. Cryo-TEM, which operates on vitrified solvent can be used to circumvent this problem.
1.5 Applications of single-chain polymer nanoparticles
Several proof-of-concept investigations have been carried out on the possible applications of SCNPs, though none of them have thus far been scaled up due to this being a relatively new field.
Nanoparticles with a globular morphology have in particular attracted interest for bioinspired applications involving the existence of variously sized cavities within the core of the micelle structure which could serve for encapsulation of drugs for their delivery or as catalytic sites (Figure 1-7).
10
Figure 1-7 Fields of possible applications for single-chain polymer nanoparticles, reproduced from ref23
In the field of drug delivery several studies have been carried out on the capability by SCNPs of successfully encapsulating and releasing molecules of medical interest. SCNPs have been shown to have a higher binding and release effectiveness than larger nanoparticles for chiral amino-acid derivatives30. Delivery of peptide in cells has also been investigated31. Amphiphilic fluorinated copolymers of PEGMA have been shown to be non-cytotoxic and capable of protein conjugation15 and have also been studied as a means of encapsulating a fluorinated agrochemical32.
Polymer thermoresponsiveness in SCNPs may be exploited for drug delivery purposes when this temperature is close to that of the human body. Larger polymer or composite nanoparticles have long been the subject of study, though not yet resulting in a clinically viable product, in order to use the LCST of given polymer blocks to operate efficient and targeted drug release from nanocarriers29. The possibility of using the LCST of amphiphilic SCNPs for this purpose has been suggested but is relatively little explored. In one study SCNPs constituted by a copolymer of PEGMA including uracil-diamido-pyridine groups capable of crosslinking via hydrogen bonds have been shown to effectively encapsulate a hydrophilic anticancer drug and being capable of effective release triggered by temperature near or above LCST (44 °C) or pH33.
Introduction
11
Another possible application in nanomedicine that has been investigated is the use as image contrast agents which has been studied for magnetic resonance imaging, tomography and fluorescence imaging, offering higher contrast than larger nanoparticles34.
The capabilities of emulating the catalytic effectiveness of natural enzymes have been summarized23, finding evidence of catalytic activity in SCNPs mimicking the one of reductase, oxidase, aldolase and polymerase enzymes. In the first pioneering studies on the self-folding of amphiphilic random copolymers both PEGMA/BTAMA and PEGMA/RMA copolymers incorporating a ligand for Ru (II) as a comonomer were shown to maintain a unimer micelle structure encapsulating the transition metal in the hydrophobic cavity and to have catalytic activity in hydrogenations13,14. These catalytic systems have demonstrated selectivity towards more hydrophobic substrates in both hydrogenations and oxydations35.
The method of self-assembly involving neutral amphiphilic polymers in water is considered the most promising in view of a possible scale-up, since it is the only one that allows for the production in one pot of a high concentration (100 g/L dispersions have been obtained that were still stable after 4 months26) of nanoparticles in water, while many of the other discussed means of obtaining SCNPs avoid the formation of multi-chain aggregates only at very low concentration (<< 1 g/L)34.
The controlled and tailored synthesis of amphiphilic polymers is nowadays feasible by an array of chain growth polymerization reactions by means of reversible-deactivation radical methods.
1.6 Reversible-deactivation radical polymerization
In conventional radical polymerization (CRP) polymers are formed through a radical chain reaction involving the monomer and an initiator. The lifetime of the growing radical is very short (<1 s) and is the only period where individual chain growth occurs, until a termination event interrupts it. This results in a fairly broad
12
distribution of molecular weights and in “dead” polymer chains that can no longer undergo a chain reaction. Despite these limitations CRP is widely used in both industrial and laboratory polymerizations when compared to ionic chain growth polymerizations because of its compatibility with a wide range of monomers, generally low costs, possibilities of operating in an aqueous environment in its dispersion and emulsion variants as well as a degree of tolerance for trace impurities of protic solvents or oxygen.
In an effort to improve on conventional radical polymerization, it has been found that the outcome of such a polymerization can be controlled by reversible deactivation of chain carriers. A 2010 IUPAC recommendation36 proposes the
term reversible-deactivation radical polymerization (RDRP) for such processes, also commonly called controlled radical polymerizations or living radical polymerizations.
A number of different techniques have been developed in the field, including, among the most important: nitroxide-mediated polymerization (NMP), reversible addition-fragment chain-transfer (RAFT) polymerization and atom-transfer radical polymerization (ATRP) with its offshoots.
The control of chain length and polydispersity given by such techniques has resulted essential in the field of self-assembling SCNPs, making it possible to obtain low polydispersity chains of the desired length which can also be “living” and extensible with a different polymer block.
The various techniques work by a similar mechanism (Scheme 1-1). The irreversible termination events common in CRP are kept to a minimum by strongly reducing the concentration of radicals, which disfavors chain termination and chain transfer. Only a small fraction of the growing chains is in active radical form, while most of them are in a dormant form that is not directly capable of chain growth. If the interconversion rate between dormant and active species is much greater than propagation rate, all chains grow at the same rate and if the initiation step is also very fast and all chains can be considered to start growing at the same time, then very low polydispersity can be achieved.
Introduction
13
Scheme 1-1 General reversible-deactivation radical polymerization mechanism
1.6.1 Atom transfer radical polymerization (ATRP)
In atom transfer radical polymerization the dormant species is constituted by a halogen-terminated growing polymer chain. The initiator is an alkyl halide which includes some sort of radical-stabilizing group (phenyl, carboxyl). A transition metal halide catalyst where the metal is capable of oxidation states separated by a single atom is also necessary. Initial radical generation is through a redox reaction between the metal halide and the alkyl halide, the latter then starting chain growth with a monomer. Regulation of the chain reaction is provided by an equilibrium between growing radicals and the dormant species, mediated by the metal catalyst (Scheme 1-3).
Scheme 1-2 General equilibrium mechanism of ATRP
Copper is the most common transition metal used in ATRP due to low cost and ease of handling. Ruthenium is the other main metal to be used. The halogen is usually either Cl or Br. A dentate ligand favors the solubility of the metal halide in the chosen solvent and regulates redox potential of the equilibrium reaction (Scheme 1-3).
14
Scheme 1-3 Common initiators (above) and ligands (below) for ATRP
Some degree of irreversible chain termination is inevitable. However, the very existence of such processes at the initial stage of the polymerization provides additional control over polydispersity through a mechanism known as permanent radical effect (PRE). The termination of the growing chains reduces their concentration and drives the ATRP equilibrium further towards the dormant species.
The main disadvantages of ATRP compared to other forms of RDRP when it comes to industrial scale-up are the high sensitivity to trace impurities of oxygen and the relatively large quantity of transition metal catalyst that needs to be used. As irreversible termination does occur during the initial stages of the reaction, leading to PRE, the catalyst concentration cannot be too low to avoid all catalyst being consumed during this stage. The common concentration used is equimolar with the initiator and leads to potentially costly purification of the catalyst from the polymer that still risks of traces of the metal being found in the final product.
1.6.1.1 Activators regenerated by electron transfer (ARGET)
Variants of ATRP have been developed which minimize the quantity of transition metal used bringing its final concentration to ppm level. ARGET is one of the main such techniques. The copper catalyst that is oxidized by eventual termination
Introduction
15
events and that would be inactive in ordinary ATRP is reduced and so regenerated by a reducing agent, various of which have been used for the purpose, including among the most common ascorbic acid and tin(II) 2-ethylhexanoate (Scheme 1-4).
Scheme 1-4 Mechanism for ARGET-ATRP, reproduced from ref37
.
The significantly lower quantity of transition metal catalyst used (generally around 1% compared to ATRP) makes its purification from the polymer unnecessary for some uses. The system also has a higher tolerance for oxygen compared to ATRP. The copper catalyst is introduced directly in its higher oxidation state and reduced in situ.
1.6.1.2 Cu(0)-mediated RDRP
A further variant of ATRP is constituted by the use of zero-valent metal. This technique is variously known as single electron transfer living radical polymerization (SET-LRP) or supplemental activator and reducing agent ATRP (SARA-ATRP) depending on the envisioned mechanism. In the former Cu(0) acts as the major activator of alkyl halides, in the latter it is CuBr. It has attracted interest due to the mild conditions used and the easy separation and reuse of the catalyst (Figure 1-8).
16
Figure 1-8 Proposed mechanisms of Cu(0)-mediated RDRP
Reversible-deactivation radical polymerizations can be employed for the synthesis of a diverse range of polymers. Among these it has been used for fluorinated polymers, including fluorinated (meth)acrylates and styrenes.
1.7 Properties of fluorinated polymers
Fluorinated polymers have many important distinctive and attractive properties, like high thermal and chemical resistance. Other features of fluorinated polymers are their low flammability, resistance to oxidation and hydrolytic decomposition, low refractive index and low dielectric constant. In addition, surface properties such as low wettability, anti-sticking properties, low friction coefficient and low adhesion in addition to hydro- and lipophobicity are generally present in polymers with a high density of C–F bonds.
1.7.1 Pentafluorostyrene-based polymers
Pentafluorostyrene (PFS) is a fluorinated aromatic monomer. Industrial interest in the applications of pentafluorostyrene-based polymers has been foremost in the field of waveguides, owing to its properties as a low optical loss material to
Introduction
17
be employed as a thin film. For such purposes it is used as a block copolymer with other monomers because of the poor solubility of the homopolymer38. In the field of non-amphiphilic SCNPs, poly-PFS blocks have been studied in a RAFT-synthesized block copolymer between pentafluorostyrene and styrene based on the π–π interaction between their aromatic rings, exploiting the electron-deficient character of the fluorinated aromatic ring to create intramolecular interactions6. Multichain conventional amphiphilic micelles based
on poly-PFS blocks have also been synthesized by creating a block copolymer with PEGMA via ATRP. These systems self-assemble in water in brush-type aggregates with PFS blocks forming the hydrophobic core and PEGMA blocks extending outwards in 20-60 nm micelles, each made up of 25-34 copolymer chains39. Surface coatings created from these block copolymers exhibited phase separation and antibiofouling properties40. Random copolymers of PFS and PEGMA have also been created via NMP for the purpose of lowering the LCST of poly-PEGMA41. The self-assembly of similar random copolymers synthesized via conventional radical polymerization was studied and, compared to the block copolymers, they were found to form quite small micelles, in the 10-20 nm diameter range, and to also exhibit a LCST tunable by copolymer composition, above which the small micelles transitioned to larger micelle aggregates, with the hydrodynamic diameter of these aggregates capable of a large dimensional range (100-1200 nm) depending on monomer ratio (Figure 1-9)42.
18
As a monomer PFS is particularly well suited to modification by nucleophilic substitution, oriented preferentially to the para position, which can result in a wide variety of functional groups being attached. The polymerization of pentafluorostyrene and its para-substituted monomers by ATRP methods has been reviewed and the base monomers were found to be as versatile as styrene, when it comes to polymerization conditions, and to contain vast potential for the creation of new materials when substituted43.
The use of triethylene glycol as one such substituent has been thus far explored for the purposes of creating a material with anti-biofouling properties. A hyperbranched amphiphilic ATRP-polymerized system featuring this substitution was first synthesized and characterized in 200744,45. The hyperbranched polymer was found to be effective at forming an antibiofouling surface while also incorporating noradrenaline as an active fouling deterrent46. The homopolymer of triethylene glycol-substituted pentafluorostyrene has been synthesized via ATRP and been used as part of a block copolymer with poly(dimethyl siloxane) finding that such a system is capable of undergoing comprehensive surface reconstruction after immersion in water47. Block copolymers with PDMS-modified styrene also displayed anti-biofouling properties48.
1.8 Properties of oxyethylenic polymers
Polyethylene glycol (PEG) is the polyether obtained from ethylene oxide. The C-O bond confers the chain a high mobility and as a result the glass transition temperatures of the polymers tend to be quite low, in the -50 to -20 °C range. In common terminology PEG is generally used to refer to oligomers and low molecular weight (<20000) polymers while poly-ethylene oxide (PEO) is used for higher polymers49. PEG may be hydroxyl- or alkoxy-terminated on either end, depending on the initiator used for the ring-opening polymerization of ethylene oxide and the termination agent, a common case being a polymer methoxy-terminated on one end (mPEG). PEG is soluble in water and highly hygroscopic due to its capability of forming hydrogen bonding with water.
Introduction
19
It is generally considered biologically inert and safe leading to a widespread use in the medical field. Incorporation of PEG into pharmaceutical formulations, achieved by various means (covalent and non-covalent attachment as well as blending), can be used to provide water solubility to otherwise insoluble drugs as well as mask them from the immune system. For these reasons the widespread use of PEG is one of the main features of the modern pharmaceutical industry50.
20
2 Objectives of the work
Amphiphilic polymers possess multimode capacities of self-assembly on nano-to-micrometer length scales when present in a variety of states, e.g., from dilute solution to thin film. Amphiphilic random copolymers can self-fold in single-chain nanoassemblies, so-called unimer micelles, via the intramolecular interactions of the hydrophobic component. This idea finds inspiration from the extremely precise folding process typical of natural polymers. These, from a given primary structure, can convey complex functions, strictly related to their secondary and tertiary structures.
Synthetic polymer self-assembly is still far from nature perfection. Nevertheless, a tailored synthesis and in-depth characterization of size, shape, core-shell structure and conformation of self-folding polymers might drive to their future exploitation as novel functional nanospaces for advanced applications in, e. g., nanomedicine and nanocatalysis. Functional random copolymers effectively allow for an intramolecular association of the functional pendants via physical interactions, including host−guest, coordination and hydrogen bonding, while they can also undergo the intramolecular cross-linking of the pendants via covalent bond formation.
One of the most recent and straightforward approaches to single-chain self-folding is the intramolecular self-assembly of well-designed amphiphilic random copolymers driven by hydrophobic interactions in a selective solvent, notably water. Typical precursors of the hydrophilic component of single-chain nanoassemblies have been reported to be either ionic monomers, such as 2-acrylamido-2-methylpropanesulfonate12,51, or non-ionic monomers, such as poly(ethylene glycol) methacrylate (PEGMA)15,24 or acrylamide (PEGAAm)20. Random copolymers based on the latter type of monomers have the advantage to be soluble in both water and organic solvents and to be thermally responsive in water solution, due to the occurrence of a lower critical solution temperature-type (LCST) transition. On the other hand, typical precursor of the hydrophobic components are fluorinated(meth)acrylates15,17,19,52, polysiloxane (meth)acrylates19 or alkyl(meth)acrylates16.
Objectives of the work
21
Among fluorinated monomers, pentafluorostyrene (PFS) presents the possibility of a facile nucleophilic substitution of the para-fluorine, making PFS a versatile building block to provide further functionalities to the resulting polymers. To the best of our knowledge no amphiphilic homopolymers have been investigated as systems intrinsically able to self-fold in a selective solvent.
Accordingly, the ultimate aim of this work was to synthesize amphiphilic homopolymers capable of reversibly self-folding in dynamic single-chain nanocompartments, which can self-assemble in multichain nanostructures as a response to an external stimulus, i.e. temperature, according to a LCST-type phase separation. Specifically, tetrafluorostyrene monomers (EFSn) carrying in para position an oligoethylene glycol chain with different lengths (n = 3, 4, 8 and 13) (Scheme 2-1) were to be synthesized and used for the preparation of the corresponding amphiphilic homopolymers (pEFSn-x) by ARGET-ATRP. By taking advantage of the controlled nature of such a polymerization technique, it was intended to vary the number average degree of polymerization (x) in order to obtain homopolymers with different values of x for a given value of n. If, on one hand, increasing the length of the oligoethylene glycol side chain (n) was anticipated to increase the hydrophilic/hydrophobic balance of the homopolymer and affect both its solubility in water and self-association behavior in solution, on the other hand the variation of the number average polymerization degree was expected to play a role in determining the size and shape of the nanoaggregates in solution.
Scheme 2-1 Structure of 4-(oligo ethyleneglycol monomethyl ether)-2,3,5,6-tetrafluorostyrene monomers (EFSn) and their ATRP-polymerized homopolymers (pEFSn-x)
22
The self-assembly behavior of the amphiphilic homopolymers in solution was to be investigated by DLS in organic solvents (e.g., CHCl3, THF, DMF, trifluorotoluene) and in water at room temperature and as a function of temperature to study the polymer thermoresponsiveness and its dependence on the chemical structure, number average polymerization degree and in terms of length of the oxyethylenic side chain. Such investigations could be complemented by small angle X-ray scattering (SAXS) to determine the size and morphology of single chain nanostructures.
The ability and effectiveness of such homopolymers to self-assemble in the bulk and at the surface of thin films was also regarded as worth studying by differential scanning calorimetry (DSC) and X-ray photoelectron spectroscopy (XPS) analyses, respectively. In fact, as a result of their susceptibility to self-aggregate in solution, microphase separate in bulk and self-assemble at the surface of thin films, the amphiphilic homopolymers of this work may be exploitable in a wide range of advanced applications, going from nanovehicles for hydrophobic drugs and nanoreactors, to protein or enzyme bioconjugates up to surface modifiers for coatings and textiles.
Results and discussion
23
3 Results and discussion
New amphiphilic monomers derived from pentafluorostyrene and poly-ethylene glycol were synthesized. The homopolymers derived from the monomers were prepared via reversible-deactivation radical polymerization, with the aim of preparing materials with a varied range of degrees of polymerization. The polymers were mostly studied in solution, with the aim of comparing their behavior with the one typical of amphiphilic fluorinated random copolymers, which are capable of self-assembly into single chain nanoparticles and have thermoresponsive characteristics. Bulk characterization of some thermal and surface properties of the polymers was also carried out.
3.1 Monomer synthesis
The amphiphilic monomers chosen for this work consisted of two distinct chemical components.
Pentafluorostyrene (PFS) was chosen as the hydrophobic part of the monomers on the basis of its random copolymers with hydrophilic comonomers being capable of self-assembly into small-size structures with thermoresponsive properties in water42.
A series of commercially available monomethyl ether oligoethylene glycols (mPEG) with degrees of polymerization (n) ranging from 3 to 13 provided the hydrophilic part of the monomers, a list of the glycols and their effective chain length measured by NMR is provided (Table 3-1).
24
Table 3-1 Used monomethyl ether oligoethylene glycols Glycol Mn declared by the supplier (g/mol) Average n declared by the supplier Determined na Determined Mn (g/mol) mPEG3 164 3.3 3.1 154 mPEG4 208 4.3 4.2 202 mPEG8 350 7.5 8.2 378 mPEG13 550 12.1 13.1 594 a By 1H NMR
A previous synthesis of EFS3 first carried out in 200744 formed the reference for the synthesis of each monomer. A nucleophilic aromatic substitution reaction of the para fluorine atom of PFS with the chosen monomethyl ether glycol (mPEGn) was conducted using sodium hydride as the base. However, at the reported reaction temperature of THF reflux (66 °C) severe formation of by-products was noticed for both the EFS4 and EFS13 syntheses. Conducting reactions at 0 °C proved effective in all four cases at reducing by-products to a minimum. Reaction times in the original recipes were found to be exceedingly long for gram-scale reactions, as near-quantitative conversions were reached after at most 5 hours. The reagent ratio used was 1/1.5/1.2 mPEGn/PFS/NaH (Scheme 3-1).
Results and discussion
25
Purification of the reaction product was performed by different methods for each monomer. In all cases eventual unreacted monomethyl ether glycol was removed through repeated washings of water solutions with ethyl acetate. EFS8 and EFS13 proved unwieldy to pass through silica gel chromatography columns. Therefore, given the very limited formation of by-products in the reactions, it was decided not to proceed for further purification. Mineral oil present as a residue from the dispersion of sodium hydride was eliminated by washing an aqueous solution of the monomers with petroleum ether. Residual traces of inhibitor were removed via a final elution on a neutral alumina column. Residual PFS was removed by prolonged drying under vacuum.
Figure 3-1 1H NMR spectrum of EFS4, to be taken as general reference for peaks of all monomers
In the syntheses of EFS4 and EFS3, purification of the product through silica gel flash chromatography was carried out after removal of the unreacted glycol. As no visible reduction of by-product traces by chromatography was observed via NMR, the main role of the chromatography was essentially the removal of mineral oil. In later syntheses the chromatography step was bypassed, as it was found that the raw product was polymerization grade itself, and indeed no adverse
26
effects on monomer conversion or polydispersity were noticed. In these cases the final precipitation of the polymer in n-hexane after polymerization served to eliminate residual mineral oil.
1H and 19F NMR analyses were used to verify the effective synthesis of the desired monomers occurred. 19F NMR analysis in particular revealed the absence of peaks other than the ones attributable to the fluorine atoms in meta and ortho positions relative to the double bond; in addition, their double doublet multiplicity showed that the nucleophilic substitution occurred exclusively on the para fluorine of PFS (Figure 3-2). This stands in contrast with reactions carried out at higher temperatures, which showed instead a higher number of peaks with different multiplicities, owing to multiple non-selective substitution on the phenyl ring.
Figure 3-2 19F NMR spectrum of EFS3, to be taken as general reference for peaks of all monomers
3.2 Polymer synthesis
Results and discussion
27
The original procedure elaborated by Matyjaszewski and colleagues was taken as reference37. CuBr2, (1-Bromoethyl)benzene (1-BEB), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDTA) and tin(II) 2-ethylhexanoate (Sn(EH)2) were chosen as metal catalyst, initiator, ligand and reducing agent respectively as they are all among the most commonly used for the purpose. The presence of the phenyl ring on the initiator could also enable us to estimate degree of polymerization. A ratio of x/1/0.1/0.01 between monomer/initiator/reducing agent/copper(II) catalyst is classically used in ARGET, x being the target degree of polymerization at full conversion, with the ligand being used in the appropriate molar proportion to both copper(II) catalyst and reducing agent, in the case of PMDTA being equimolar to both. We used a greater proportion of metal catalyst, with the CuBr2/initiator ratio ranging from 0.05 to 0.03. As the CuBr2/PMDTA complex proved incompletely soluble in anisole at room temperature the metal catalyst was added either by direct weighing or by using a solution of the complex in methanol followed by complete removal under vacuum of the polar solvent. The polymerization reaction was then carried out at 110°C, under nitrogen atmosphere or vacuum (Scheme 3-2).
Scheme 3-2 Synthesis of pEFS polymers
To study the effect of polymer chain length on self-assembly we prepared for each monomer a series of samples with different degrees of polymerization including “short” (<20 repeating units), “medium” (20-40 units) and “long” (>40 units) chains. As the pEFS8 polymer turned out to be the one of greater interest,
28
additional samples with longer macromolecular chains (≤ 135 units) of this polymer were also synthesized.
The different polymer samples are identified as pEFSn-x where n (3-13) is the average degree of polymerization of the mPEG side chain and x is the average degree of polymerization of the polymer (8-135).
ARGET-ATRP polymerization under nitrogen atmosphere ultimately proved unreliable in obtaining effective conversion up to a significant degree (>50%) of the monomer, despite ARGET being commonly considered less susceptible to trace oxygen than normal ATRP. Most of the polymerizations were therefore conducted under vacuum. In these cases, quantitative monomer conversion was achieved within 24 hours in almost all reactions, the exception being the highest monomer/initiator ratio (200).
Cu(0)-mediated RDRP was also attempted on PFS to obtain a PFS homopolymer as a reference polymer. The resulting polymer was found to be quite polydisperse (Mw/Mn = 2.22) and the conditions chosen were considered not amenable to a controlled polymerization.
Results and discussion
29
A full table of polymerization results for ARGET-ATRP is provided (Table 3-2). Table 3-2 List of synthesized pEFSn-x polymers
Polymer Atmo- sphere Monomer/ initiator ratioa Conversion (%) x Mnb (g/mol) Mnc (g/mol) 𝑀𝑤 𝑀𝑛 pEFS3-17 N2 25 55 17 5750 4400 1.14 pEFS3-28 N2 50 50 28 9470 6400 1.23 pEFS3-46 Vacuum 60 92 46 15560 10700 1.23 pEFS4-17 Vacuum 15 90 17 6360 6000 1.15 pEFS4-22 Vacuum 30 83 22 8230 6100 1.18 pEFS4-53 Vacuum 60 94 53 19830 13000 1.28 pEFS8-16 Vacuum 15 95 16 8870 9800 1.17 pEFS8-26 N2 24 99 26 14410 10800 1.26 pEFS8-46 Vacuum 60 93 46 25490 19800 1.25 pEFS8-72 Vacuum 200 60 72 39900 34700 1.31 pEFS8-135 Vacuum 120 90 135 74800 35800 1.53 pEFS13-8 Vacuum 6 95 8 6150 9100 1.42 pEFS13-25 Vacuum 25 96 25 19200 15100 1.37 pEFS13-46 Vacuum 60 90 46 35330 30200 2.30 pEFS13-54 Vacuum 60 85 54 41480 23400 1.75 a Mol/mol ratio b By 1H NMR c By GPC
Conversion was estimated by analyzing a sample of the quenched reaction mixture by 1H NMR and comparing the integral of the signal of the double bond NMR signal at 5.65 ppm with the integral of the terminal methyl group of the oxyethylenic side chain at 3.34 ppm. The former decreased as the monomer was polymerized, while the latter remained the same throughout (Figure 3-3).
The average degree of polymerization was estimated by comparing the integral of the aromatic signal (6.9-7.2 ppm) of the phenyl ring associated with the initiator residue on one terminal of the polymeric chain with the integral of the terminal methyl group of the oxyethylenic side chain. Deuterated acetone was used as the
30
solvent for purified polymer NMR analysis, with an added portion of deuterated chloroform (1/3 in volume compared to acetone) to improve signal quality. Using acetone as the NMR solvent resulted in a splitting by the Ar-O-CH2 peak located
at 4.2-4.4 ppm, which was not detected in similar conditions in chloroform. Number average molecular weight (Mn) was then calculated both by this measure of degree of polymerization and by a gel permeation chromatography (GPC) measurement (Figure 3-4). The latter value is expected to be less accurate, as GPC needs a series of monodisperse polymer standards to obtain a calibration fit and the standards used (polystyrene) are different in conformational and hydrodynamic characteristics from the obtained polymers. The polydispersity index (PDI) of the polymer was also calculated by GPC.
6 8 10 12 14 16 18 20 0,0 0,2 0,4 0,6 0,8 1,0 16 26 46 72 Int en sity (n or ma lized) Time (min) Degree of polymerization
Figure 3-4 GPC elution curves of pEFS8-x at different degrees of polymerization
Most of the polymers were obtained at a degree of polymerization consistent with the combination of monomer/initiator ratio and conversion, with quite low PDI
Results and discussion
31
(1.1-1.3). Two main exceptions were noted: at the highest monomer/initiator ratios used for pEFS8 both polydispersity and the difference between expected and measured degree of polymerization increased. This may be caused by a combination of termination control by the ATRP equilibrium being weaker with less metal catalyst being present and growing integration error on the NMR signals as the initiator peaks get smaller. Polymers synthesized from EFS13 also showed significantly higher polydispersity than the ones synthesized from other monomers, a finding which was especially noticeable in the attempts to obtain the long chain pEFS13 polymers. It can be hypothesized that this polymerization reaction suffers much more than the others from irreversible termination at high conversions, probably by combination as the GPC curve shows a bulge towards high molecular weights. As polydispersity is known53 to trend down with conversion, the polymers might be obtained with acceptable molecular weight control by stopping the reactions at lower conversions (<80%).
Another tool to characterize the product of the controlled polymerization would be the presence in most NMR spectra of our polymers of a peak located at 5.0-5.2 ppm, attributed to the terminal, bromine-bonded, -CH group. However, this peak was not visible in the more polydisperse polymers, which might indicate a high ratio of irreversible termination, though the peak was less visible in the spectra of higher molecular weight polymers due to its low relative size.
3.3 Solubility properties of amphiphilic monomers and polymers
The solubility in water at room temperature of EFSn monomers and pEFSn-x polymers was tested. For substances insoluble in water we then tried to find the least proportion of a less polar water-miscible cosolvent (methanol) which led to full solubility of the chosen sample. The results are reported in Table 3-3.
32
Table 3-3 Solubility properties in water of EFS monomers and their polymers
Water soluble Methanol proportion (volume %) Monomer EFS3 40 EFS4 35 EFS8 EFS13 Polymer pEFS3-x 50-55 pEFS4-x 25-30 pEFS8-x pEFS13 -x
Only the monomers with longer oxyethylenic chains, as well as their respective polymers were fully soluble in water. All samples proved soluble in methanol and required different proportions of methanol as a cosolvent with water to become soluble in a water/methanol mixture. Such a solution was considered the most polar medium in which these substances could be solvated and was as such later used to study their self-assembly, as water alone was not an option.
The quantity of methanol needed to achieve solubility was found to decrease with increasing oxyethylenic chain length, since longer chains bring higher hydrophilicity. While polymers of EFS3 needed a higher methanol proportion than the corresponding monomer to become soluble, the opposite was true for polymers of EFS4, which proved more hydrophilic than the corresponding monomer.
The critical micelle concentration (CMC) of water-soluble monomers was also estimated, on account of their expected surfactant-like properties. CMC can be evaluated by dynamic light scattering (DLS) analysis by measuring the intensity of scattered light and plotting it against the logarithm of concentration. Two different possible linear fits should be present at low and high concentrations (surfactant forming a layer on the surface and micelles in the bulk of the dispersant respectively), with their intercept corresponding to the CMC (example in Figure 3-5).
Results and discussion
33
Figure 3-5 Estimation of critical micelle concentration for EFS13 via dynamic light scattering intensity measurements
The evaluated CMC of the two monomers is reported in Table 3-4. Table 3-4 Critical micelle concentration for water-soluble monomers
Monomer CMC (g/L)
EFS8 1.0
EFS13 1.2
3.4 Thermal properties of amphiphilic monomers and polymers
in solution
It is common for amphiphilic polymers to display thermoresponsive solubility behavior in water with a lower critical solution temperature (LCST) behavior, as the polymer chain changes its behavior at higher temperatures by losing its hydrogen bonds to water and expelling its bound water into the bulk, creating inter-macromolecular bonds instead. The transition can be easily distinguished by the naked eye, as it results in a cloudy dispersion of the resulting aggregates instead of a clear or mostly clear solution (Figure 3-6). This macroscopic change means that a transmittance measurement at a wavelength in the visible light can
34
be used for a facile determination of cloud point temperature (Cp). We used an incident visible light at 700 nm wavelength and set the Cp as the temperature at which transmittance decreased to 50%.
Figure 3-6 Pictures of pEFS4-53 in a 4/1 v/v water/methanol mixture below (left) and above (right) the cloud point temperature
All synthesized polymers and both water-soluble monomers displayed such a thermoresponsive behavior, both in water and in a water/methanol mixture, whereas the EFS3 and EFS4 monomers did not. If the unimer micelle structure of the polymers in solution is accepted, the loss of bound water does not happen throughout the polymer chain as it does in macromolecules which take a random coil conformation in water (Figure 3-7), but on the hydrophilic shell of the core-shell micelle structure, resulting in the micelles aggregating in a grape-like manner.
Results and discussion
35
The water-soluble monomers do not form unimers in solution, but probably arrange themselves as typical surfactants in classical multimolecular micelles (see DLS dimensional analysis below). Their aggregation is still expected to proceed in a similar manner to unimer micelles, with water bound to the hydrophilic PEG shell being expelled above the LCST and multi-micelle aggregates forming.
The behavior noticed by the two monomers in proximity to LCST was quite distinct. For EFS8 the change was very sharp as Cp (33°C) was reached, while EFS13 showed a progressive clouding of the initial solution in a temperature interval of about 25 °C, where Cp was estimated to be 54°C by taking the point corresponding to 50% transmittance (Figure 3-8).
Figure 3-8 Transmittance of light at 700 nm of 5 g/L solutions of water-soluble monomers
Synthesized polymers exhibited a sharp LCST-like transition to cloudy dispersions in all cases, whether dissolved in water and a water-methanol mixture. Some difference (in a range between 2 and 9 °C depending on the polymer) was noticed in the exact location of Cp of the same polymers at different degrees of polymerization but there was no clear trend where Cp varied along with chain length. Full graphs of transmittance for pEFS8 are reproduced as illustration examples (Figure 3-9), the other polymers also exhibited a similar sharp transition. A full list of cloud points is also reported (Table 3-5).
0 10 20 30 40 50 60 70 80 90 100 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 Tran sm itta n ce (% ) Temperature (°C) EFS8 EFS13
36
Results and discussion
37
Table 3-5 Cloud points of polymer solutions (5 g/L in water unless otherwise noted), 50% transmittance taken as cloud point
Polymer Cp (°C) pEFS3-17 38a pEFS3-28 39b pEFS3-46 37b pEFS4-17 31c pEFS4-22 34d pEFS4-53 38d pEFS8-16 72 pEFS8-26 69 pEFS8-46 72 pEFS8-72 78 pEFS8-135 73 pEFS13-8 86 pEFS13-25 92 pEFS13-46 85
a 55% (volume) methanol as cosolvent b 50% (volume) methanol as cosolvent c 30% (volume) methanol as cosolvent d 25% (volume) methanol as cosolvent
A trend was noticed in water-soluble polymers, for which the thermal transition happened at significantly higher temperatures than in the corresponding monomers. In both monomers and polymers, a longer PEG side chain corresponded to a higher Cp. This is something that is analogous to the behavior of PEGMA polymers with varying side chain length28. As the hydrophilic portion of the polymer gets longer, stronger hydrogen bonds with water are formed and a higher temperature is required to break them. If this stays true, it is expected that pentafluorostyrene polymers with longer oxyethylenic side chains will not show a LCST in water at all at standard pressure.
38
The lower transition temperatures shown by the non-water-soluble polymers in water/methanol mixtures also seems to confirm this trend, although the different solvent used makes direct comparison impossible, as its hydrogen bonding character gets weaker as the methanol proportion increases.
3.5 Single-chain folding and self-assembly of amphiphilic
polymers in solution
Dynamic light scattering (DLS) experiments on amphiphilic pEFS homopolymers and their monomers were carried out to evaluate their ability to spontaneously single-chain fold in a suitable solvent, like water or a water-methanol mixture, forming unimer micelles driven by the association of hydrophobic portions of the monomeric unit. Small-angle X-ray scattering (SAXS) measurements in water were also carried out to investigate the effective structure of the polymers in solution.
3.5.1 Dynamic light scattering of EFSn and pEFSn-x solutions
For every sample, 5 g/L (if not otherwise stated) solutions in water or other organic solvents were prepared by dissolution in 0.2 μm filtered solvents of the highest purity available. Solutions were analyzed at 25°C and at a temperature above the Cp previously evaluated.pEFS13 polymers were not analyzed above Cp because of instrumental temperature limitations. At least 5 separate measurements were carried out for each solution. When all the results were coherent with each other an instrument software-created average size distribution curve was considered as an overall representative of the system. In addition to the polymers, the water-soluble monomers (EFS8 and EFS13) were analyzed. Both were found to form particles with a hydrodynamic diameter (Dh) of several hundred nanometers, significantly larger for EFS13 compared to EFS8 (Figure 3-10 and Table 3-6). This confirms the formation of classical aggregate micelles, with a hydrophobic core constituted by the hydrophobic “heads” and a
Results and discussion
39
hydrophilic shell constituted by the hydrophilic “tails” of the molecules. Past Cp both monomers were found to form micron-sized aggregates.
Table 3-6 DLS size distribution data for water-soluble monomers
Monomer EFS8 EFS13
Hydrodynamic diameter (nm) at 25°C 160 ± 60 500 ± 200 Hydrodynamic diameter (nm) above Cp 1300 ± 500 1100 ± 200
Figure 3-10 DLS intensity size distribution for water-soluble monomer, 5 g/L solution in water at 25°C (left) and at 65°C (right)
When the pEFS polymers were analyzed, both in water and water/methanol mixture, it was found that the intensity distribution was in all cases bimodal at room temperature, with one peak indicating a smaller hydrodynamic diameter (generally less than 10 nm) and a second one a larger diameter (hundreds of nm) (Figure 3-11, right). The presence of the population at smaller diameters was consistent with the formation of folded macromolecular chains. The presence of larger diameters was ascribed to the formation, by a small minority of polymer chains, of multichain aggregates, which were difficult to dissolve at a molecular level just by mechanically stirring the solution. From the volume distribution, it is evident that the predominant form in solution is the folded unimer micelle, as the smaller diameter peaks always accounted for at least 98% of particle volume (Figure 3-11, left), notwithstanding the intensity distribution, on account of the dependence of intensity I to the sixth power of diameter, according to the Rayleigh scattering law:
0 5 10 15 20 1 10 100 1000 10000 In ten sity d is trib u tio n Hydrodynamic diameter (nm) EFS13 EFS8 0 10 20 30 1 10 100 1000 10000 In ten sity d is trib u tio n Hydrodynamic diameter (nm) EFS13 EFS8
40 𝐼 = 𝐼01 + cos 2𝜃 2𝑅2 ( 2𝜋 𝜆 ) 4 (𝑛 2− 1 𝑛2+ 2) 2 (𝐷ℎ 2) 6
where R is the distance to the particle, n the refractive index, 𝜆 the wavelength and 𝜃 the scattering angle.
Figure 3-11 DLS volume (left) and intensity (right) distribution for pEFS8-72, 5 g/L solution in water at 25°C
The reported diameter in these cases will in general be assumed to be the one from the peak which accounted for more than 98% of the volume distribution. The hydrodynamic diameter calculated by instrumental algorithms for this peak from intensity distribution was considered to be more reliable than the one calculated from volume distribution (generally smaller) and is the one reported. This phenomenon has been reported multiple times for amphiphilic random polymers containing fluorous elements15,17.
When measured at temperatures above Cp,particle diameter was in all cases above 800 nm, confirming the aggregation of polymer chains into much larger structures due to hydrophilic interactions with water not being capable any longer of solvating single unimer micelles and hydrophobic interactions becoming dominant. These aggregates were generally found to have a diameter between 800 and 1800 nm with a monomodal distribution. However, in several cases a further aggregation behavior was noticed, with even larger particles with diameters greater than 2 μm appearing in a bimodal distribution or even generating the only signal in a new monomodal distribution. This is likely caused
1 10 100 1000 10000 Volu m e d is trib u tio n Hydrodynamic diameter (nm) 1 10 100 1000 10000 In ten sity d is trib u tio n Hydrodynamic diameter (nm)
Results and discussion
41
by single polymer macroaggregate particles further coalescing with each other. A case where the phenomenon was particularly evident was pEFS8-135, where particle diameter at a temperature above Cp (80°C) was noticed to evolve from 1800 nm, in measures taken from 5 to 15 minutes after reaching said temperature, to 2600 nm in measures taken 20 to 30 minutes afterwards (Figure 3-12).
Figure 3-12 Evolution of intensity distribution of pEFS8-135, 5 g/L solution in water at 80°C
In other cases where particle coalescence occurred, however, there was no such clear evolution and particle size exhibited a multimodal distribution for the whole time when the measurements were carried out. No clear correlation between polymer structure and whether particle coalescence occurred was noticed. A full list of hydrodynamic diameters at room temperature and above Cp is available in Table 3-7. 1 10 100 1000 10000 In ten sity d is trib u tio n Hydrodynamic diameter (nm)
Measures 20-30 min after transition Measures 5-15 min after transition
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Table 3-7 Average hydrodynamic diameters from intensity distribution of aqueous solution of the pEFS amphiphilic polymers, derived from DLS intensity size distributions at 5 g/L in water unless otherwise noted
Polymer Mn (g/mol) Mw/Mn Dh at 25°C (nm) Dh above Cp (nm) pEFS3-17 5750 1.14 5±1a 800±100b pEFS3-28 9470 1.23 5±1c 1800±700b pEFS3-46 15560 1.23 7±2c Multimodalb pEFS4-17 6360 1.15 7±2d 900±300e pEFS4-22 8230 1.18 7±1f 1200±300e pEFS4-53 19820 1.28 6±2f 1100±300e pEFS8-16 8870 1.17 5.5±0.9 1800±300g pEFS8-26 14410 1.26 6±1 Multimodalg pEFS8-46 25490 1.25 7±2 1500±300g pEFS8-72 39900 1.31 10±2 Multimodalg pEFS8-135 74800 1.53 11±4 1800±400 to 2600±50g pEFS13-8 6150 1.42 6±1 - pEFS13-25 19200 1.37 7±2 - pEFS13-54 41480 1.75 12±5 - pEFS13-46 35330 2.3 30±20 -
a 55% (volume) methanol as cosolvent b T = 50°C
c 50% (volume) methanol as cosolvent d 30% (volume) methanol as cosolvent e T = 45°C
f 25% (volume) methanol as cosolvent g T = 80°C
The polymers were generally found to have a Dh comprised between 5 and 10 nm, with the exception of the more polydisperse, higher molecular weight ones. The measured Dh is compatible with diameters in other studies attributed to unimer micelles formed by the self-assembly in water of amphiphilic PEG-containing random copolymers15,16,19. Self-assembly in water/methanol mixture for water-insoluble polymers was found to lead to similarly sized particles,
