Self-assembling behaviors of amphiphilic polymers: From unimer micelles in solutions to nanostructured surfaces in thin films

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PhD thesis

Self-assembling behaviors of

amphiphilic polymers:

From unimer micelles in solutions to

nanostructured surfaces in thin films

Candidate: Elena Masotti

Supervisors:

Prof. Giancarlo Galli Dr. Elisa Martinelli

Coordinator:

Prof. Gennaro Pescitelli

PhD IN CHEMISTRY AND MATERIALS SCIENCE

XXXIII cycle (2018/2020)

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i

Acknowledgments

I would like to express my sincere gratitude to my supervisors: Prof. G. Galli and Prof. E. Martinelli for their patience, advice and time devoted to my scientific growth. During the years, solid collaborations were developed with Italian and foreign Institutions. Prof. A. Pucci and Prof. T. Biver (University of Pisa) helped with the fluorescence emission and light transmission measurements. Prof. A. Glisenti (University of Padua) assisted with the XPS analysis. Dr. C. Sissa (University of Parma) assisted in the investigations of fluorescence emission anisotropy. Prof. A. Tavanti (University of Pisa) performed biological tests. Prof. G. Paradossi and Dr. F. Domenici (University of Rome Tor Vergata) closely supported our work in SANS measurements.

Special thanks must be to Prof. F. Uhlig and his research group at the Graz University of Technology for welcoming me with kindness and helpfulness. In particular, I have to thank Dr. M. Kriechabaum for his invaluable help in SAXS measurements. I really need to thank Dr. E. Guazzelli for always having an encouraging word or advice even in the most difficult days.

Many thanks to Giuseppe Pisano, Marco Turriani, Matteo Calosi and Ilenia Fiaschi for their contribution in the preparation and characterization of the materials of this PhD work.

Finally, I want to thank all my friends, my family and Matteo who have been by my side in these three years, supporting me in all my choices.

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iii

Abstract

The PhD work was aimed to investigate novel types of amphiphilic synthetic polymers, where hydrophobic intramolecular interactions drive self-assembling into nanostructures in water solution and at the surface of thin solid films. In particular, attention was focused on the understanding of the morphology of self-assembled structures in relation to the nature of the hydrophobic component in the different polymers and to the variation of the temperature and concentration. To obtain a good control on the (co)polymerization the ARGET-ATRP and RAFT methods were used. In this way, we succeeded in preparing (co)polymers featuring diverse chemical architectures as well as tailored and varied philic/phobic balances.

Three major classes of amphiphilic polymers were newly developed: i) Random copolymers of polyethyleneglycol methyl ether methacrylate (PEGMA) as an hydrophilic and thermoresposive component with perfluorohexylethyl acrylate (FA) or polydimethylsiloxane methacrylate (SiMA) as an hydrophobic component. Triethyleneglycol methyl ether methacrylate (TRIGMA) was also used as an alternate comonomer in order to depress hydrophilicity of the respective copolymers. ii) Homopolymers of hydrophobic tetrafluorostyrene monomers modified by inserting an oligoethylene segment with varied chain lengths. iii) Random copolymers based on hydrophilic zwitterionic phosphorylcholine methacrylate (MPC) or sulfobetaine methacrylate (MSA) with 3-(trimethoxysilyl)propyl methacrylate (PTMSi) and copolymers of an hydrophobic FA with PTMSi.

Detailed characterization of the (co)polymers by dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and small angle neutron scattering (SANS) measurements confirmed the formation of self-folded, intrachain nanostructures, or unimer micelles, for amphiphilic fluorinated copolymers of type i) in water solutions at room temperature. All polymers exhibited a reversible LCST-like behavior in water solutions, as identified by a transition temperature Tc that depended on the nature and

composition of the amphiphilic polymers. The self-folded nanostructures clustered together into interchain aggregates above Tc, that quite reversibly collapsed back to

unimer micelles on cooling below Tc. By contrast, all copolymers showed a

conventional random coil conformation in organic solvents. A similar thermoresponsive behavior of intrachain nanostructures was observed with homopolymers of type ii), whose Tc was influenced by the overall

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iv hydrophobic/hydrophilic balance, as identified by the length of poliethylene glycol side chains. Fluorescent molecular probes were used to observe the self-assembly behavior of fluorinated amphiphilic copolymers. In particular, the single chain folding process was investigated through both a covalent functionalization and physical dispersion of two different hydrophobic dyes (2-cyano-2-[4-[vinyl(1-1’-biphenyl)-4’-yl]vinyljulolidine (JBCF) and coumarin 153 respectively) by fluorescence emission spectroscopy. The results of these preliminary analyses suggested the possibility of using the designed fluorinated copolymers as systems for controlled loading of hydrophobic molecules and possible release in response to a change in the embedding environments.

Random copolymers of type iii) were developed in which the zwitterion monomers MPC or MSA and hydrophobic FA were combined with functional PTMSi for tethering on glass substrates, thereby creating functional supported films incorporating nanostructuring additives. Experiments by static contact angle measurements, X-ray photoelectron spectroscopy, surface zeta potential and fluorescence spectroscopy analyses were applied to prove the effective anchoring of the polymeric films on the glass substrate before and after immersion in water. Then, antifouling potential of the films was demonstrated in biological assays against the adhesion of the yeast pathogen Candida albicans, chosen as a model fungal microorganism. The fluorinated copolymers were much more able to reduce fungal adhesion than did the zwitterionic MPC and MSA polymers.

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v

This PhD thesis work was focused on the design, synthesis, characterization and initial evaluation of special properties of novel amphiphilic (co)polymers that would be capable of assembling and nanostructuring via spontaneous organization processes promoted by the folding of single macromolecular chain. One unique and most relevant feature to be investigated was the self-folding of individual polymer chains into compact nanoobjects (sometimes referred to as unimer micelles) as driven by intramolecular hydrophobic interactions in water solutions.

To the best of our knowledge, an in-depth investigation into the morphology and conformation of non-covalent, intramolecular single-chain folded nanostructures has so far not been carried in the relation to the nature and composition of the polymer in solution. Therefore, this study was started with some proof-of-concept experiments that could help enhance the basic understanding of soft matter nanostructures that are endowed with potential for application in several advanced fields. Such systems with single chain folding would be advantageous in terms of atom economy and reduced dimensions achievable compared to the present conventional nanotechnologies, e.g. as nanoreactors in green catalysis, as nanocarriers for the transport/capture of drugs/chemicals, or as interphase nanostructuring additives of anti-biofouling devices. .Moreover, the non-covalent stabilization of the self-assembly structures would entail a considerable amplification of the possible performances, owing to reversibility of the self-assembly that would therefore be chemically modulated for a “smart” response to diverse, external stimuli.

According to the above motivations, this PhD thesis work was aimed at addressing three main themes dealing with novel polymers conceived to be able of assembling and nanostructuring in different (solution, bulk) conditions: i) self-folded amphiphilic

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Introduction

2 copolymers in solutions, ii) self-assembling amphiphilic homopolymers in solutions, and iii) amphiphilic copolymers at nanostructured film surfaces. Therefore, we will sub-divide the thesis presentation into six separate chapters, with each citing their own literature references.

The first chapter will provide a relatively concise but comprehensive introduction to the main general aspects of the topics of interest of amphiphilic polymers, their syntheses by reversible-deactivation radical polymerization methods and their relevant properties for application, notably in anti-biofouling systems.

The second chapter will deal with the unimer micelles of amphiphilic random copolymers in solution, based on the driving force of the self-assembly of hydrophilic and thermoresposive poly(ethylene glycol) methacrylate and hydrophobic fluorinated or siloxane (meth)acrylates. In addition, the ability and effectiveness of such amphiphilic random copolymers to self-assemble at the surface of thin films will also be discussed.

In the third chapter, attention will devoted to amphiphilic hompolymers of tetrafluorostyrene monomers carrying polyethylene glycol side chain with different lengths that exhibited a self-assembly behavior in solution similar to that found for amphiphilic random copolymers.

In the fourth chapter, amphiphilic copolymers based on hydrophilic zwitterionic comonomers and hydrophobic fluorinated comonomers will be presented, along with the investigation of their assembly at the outer surface of thin films for anti-biofouling performance against a widespread and aggressive fungal pathogen.

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3 The main conclusions drawn from the work will be highlighted in the fifth chapter. All details of the experiments performed during synthesis and characterization will be reported in the sixth and final chapter.

1.1 Amphiphilic polymers

Amphiphilic polymers are characterized by the presence of both hydrophilic and hydrophobic constituents in the same macromolecule. The overall amphiphilic behavior depends on the nature of the functional groups and the hydrophilic/hydrophobic balance, as well as the architecture of the macromolecule. While an amphiphilic structure can be generated in homopolymers by combinations of both components in the parent single monomers, more common examples of copolymers incorporate comonomers with separate hydrophilic and hydrophobic characters. The most typical hydrophilic monomers used (Figure 1-1, right) include neutral components, e.g. 2-hydroxyethyl- and poly(ethylene glycol)- based monomers, and negatively (e.g. sulfonate and carboxylate) charged- or positively (e.g. ammonium) charged-monomers. Zwitterionic monomers are also used that consist of both oppositely charged groups. The most chosen hydrophobic monomers (Figure 1-1, left) contain alkyl, alkylphenyl and fluoroalkyl chain groups.

Figure 1-1: Common hydrophobic and hydrophilic monomers used for preparation of amphiphilic copolymers

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Introduction

4 Macromolecules containing both hydrophilic and hydrophobic parts are generally referred to as polymeric surfactants. These have greater structural complexity than low molecular weight surfactant counterparts, which can lead to different behaviors. Several natural polymers are polymeric surfactants[1]_{, such as proteins found in many}

cases as emulsion stabilizers in natural systems (e.g. casein in milk). Polysaccharides can also be classified as natural surfactants (e.g. emulsan and chitosan)[2]_{. In most}

cases, it is difficult to isolate natural polymeric surfactants, and synthetic polymeric systems are more widely investigated. The common polymeric surfactants have the ability to self-assemble in particular selective solvents. This behavior can be exploited in several potential applications such as (mini)emulsion polymerizations, coatings, biotechnology, nanotechnology, medicine, water purification and cosmetics [3-5]_{. A}

further advantage is the possibility of introducing responsive behavior to external parameters such as pH, temperature, electrolyte concentration and UV irradiation, in order to obtain smart materials[6, 7]_.

Over the years, various studies were based on understanding the influence of different architectures on the properties of amphiphilic polymers. In fact, thanks to the development of controlled polymerization methods such as NMP, ATRP and RAFT, the number of available structures increased enormously. In general, from a structural point of view, amphiphilic polymers may be divided into two main classes[8]_:

polysoaps and macrosurfactants. Homopolymers of intrinsically amphiphilic monomers are defined polysoaps. Random, alternating and segmented multiblock copolymers of hydrophilic and hydrophobic monomers also fall into this class of surfactants. The second class includes amphiphilic linear, grafted and branched block copolymers in which the hydrophilic and hydrophobic parts are well separated[9]_{. The}

different compositions of these two classes lead to a non-similar assembly behavior in water (Figure 1-2). Polysoaps can give intramolecular interactions forming unimer micelles in water solution, while macrosurfactants seem to only give intermolecular aggregation.

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5

Figure 1-2: Represent of different behavior in water solution of polysoaps (left) and macrosurfactants (right)

1.2 Self-assembling amphiphilic polymers

When an amphiphilic (co)polymer is dissolved in a selective solvent that is thermodynamically a good solvent for one component, and a precipitant for the other, the polymeric chains can self-assemble reversibly. The self-organization of amphiphilic (co)polymers is a simple method for producing structures at the nanoscale. This phenomenon can occur due to intermolecular interactions, forming macroaggregates of polymer chains in solution, or intramolecular interactions that cause single polymer chains to collapse into self-folded nanostructures, so-called unimer micelles[10]_.

In block copolymers in solution such as water, intermolecular interactions dominate[11]_{. The formation and stability of the micelles formed by the aggregation of}

several macromolecules depend on two parameters: the critical micellar concentration (CMC) and the critical micellar temperature (CMT). At concentrations above the CMC, the copolymers self-assemble into micelles orienting the hydrophilic block outwards (akin to water), while at concentration below the CMC they disintegrate (Figure 1-3). Moreover, at a temperature below the CMT there is an increase in the solubility of the copolymer and the micelles disintegrate[11]_.

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Introduction

6

Figure 1-3: Self-assembly macroaggregate of block copolymer. Adapted from ref[12]

Over the years, there has been considerable interest in the possibility of producing nanomaterials through the reversible self-assembly phenomenon and using them in the transport and release of substances, such as drugs[13]_{. These specifically}

functionalized nanomaterials could physically trap hydrophobic molecules within their internal block in water solution, while the hydrophilic shell would favor the solubility of the carrier in a physiological environment and increase its residence time. To produce polymeric micelles for such applications it is necessary to take into account their size and their ability to be nanocontainers. These factors depend on the size and structure of the block copolymer that are used. Based on this, two types of micelles can be distinguished: star-shaped, in which the outer shell is larger than the nucleus, and brush, in which on the contrary the block of the core is predominant[14]_.

Recently, there has been considerable interest in the possibility of producing nanomaterials by single-chain folding of amphiphilic random copolymers in water due to the introduction into the chemical structure of various intramolecular interactions, such as hydrophobic effect, hydrogen bonding and metal-ligand coordination[15]_{. Developing a single chain folding device, as an alternative to}

common collective aggregates, is beneficial in terms of atom economy and size reduction, both being crucial features in current nanotechnologies. Due to their unique architecture, unimolecular micelles show excellent stability regardless of the high

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7 dilution conditions and other microenvironment changes, making them particularly attractive for the design of stable micelles for specific applications[16]_{. In fact, single}

chain folding polymers can advance a number of possible applications as unimolecular objects, e.g. in catalysis and drug transport and release, or can be used for the operation of substrates, for example in microelectronics.

It is possible to design a fast, simple and versatile method for direct monitoring of spontaneous organization in solution, and perhaps in bulk, by introducing in the self-assembling system fluorescent probes, chemically linked or physically embedded, that are able to selectively interact with either the hydrophilic or hydrophobic component[17]_.

1.3 Single-chain folding polymers

In recent years several methods for the preparation of single-chain polymer nanoparticles (SNCPs), in which the polymer chain collapsed into a nanometer-size particle have been developed[18]

. Such SNCPs can be obtained by reducing the

conformational freedom of the macromolecular chain via various kinds of intramolecular interactions, both covalent and non-covalent[19, 20]_{. In literature, the first}

report dates back to 2001 with the formation of SCNPs via intramolecular crosslinking of a linear precursor containing acryloyl or methacryloyl functional, polymerizable groups[21]_{. The self-folding of such chains through the formation of covalent}

intramolecular bonds was the first option explored. For this purpose, a number of possible intramolecular covalent non-reversible crosslinking procedures were introduction as Diels-Alder (DA) reaction[22] _{in 2002, cross-metathesis}[23]_and

quinodimethane formation[24] _{in 2007, cooper(I)-catalyzed azide-alkyne cycloaddition}

(CuAAC)[25]_{and amine formation}[26] _{in 2008, benzoxazine ring opening}

polymerization (ROP)[27]_{, Bergman cyclization}[28]_{and nitrene crosslinking}[29]_{in 2011,}

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Introduction

8 ROP[33]_{in 2013, and tetrazine-norbornene reaction}[34]_{, nitrile-imine ligation}[35]_,

thiol-yne coupling[36]_{and lactone ROP in 2014}[37]_{. Details of such processes will not be}

shown since they were out of the scope of this work.

Over recent years, in addition to single-chain folding covalent systems, other approaches towards reversible self-assembling polymers were developed. Such macromolecules have a stimuli-responsive behavior more similar to that functional single-chain polymers present in natural systems. The first preparation of responsive SCNPs was in 2008 via the dimerization of benzamide through hydrogen bonding[38]

(Figure 1-4).

Figure 1-4:Schematic of a responsive SCNP preparation via chain collapsing and hydrogen bonding[38]

On the other hand, in the literature there are studies on other types of reversible crosslinking pathways, such as π-π interactions, host-guest systems, metal-ligand interactions and hydrophobic interactions.

To obtain water-soluble SCNPs, without further chemical reactions, solvent-induced self-folding was exploited. Such macromolecules have a hydrophobic part that promotes chain folding in water and a hydrophilic part, which provides solubility in water. In particular, biodegradable graft copolymers of poly(γ-glutamic acid) and L-phenylalanine represent the first macromolecules prepared to form water-soluble unimer nanoparticles by addressing the hydrophilic-hydrophobic balance of graft

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9 copolymers[39]_{. In subsequent developments, commonly used hydrophilic elements}

were oxyethylene chain segments present in monomers able of free radical polymerization, notably poly(ethylene glycol) methacrylate (PEGMA). Terashima et

al. in 2011 introduced a PEGMA based copolymer with

benzene-1,3,5-tricarboxamide methacrylate (BTAMA) as hydrophobic comonomer[40]_{. In this way,}

the self-folding in water solution was due to both hydrogen bonding and hydrophobic interactions. Subsequently, hydrophobic interactions were found to be sufficient for the formation of nanoparticles in water (diameter ~ 3-4 nm). For this reason, later studies focused on the use of methacrylates with alipathic or perfluorinated side chain of various lengths as hydrophobic comonomers. Sawamoto and collaborators[41]_in

2014 reported on the synthesis of random amphiphilic copolymers via ruthenium-catalyzed living radical polymerization of hydrophilic PEGMA and hydrophobic alkyl methacrylate (RMA), where degree of polymerization and hydrophobic R moiety were varied (Figure 1-5).

Figure 1-5: Single-chain folding of PEGMA/RMA copolymers in water solution[41]

The nanoparticles reversibly formed by the single-chain folding of such macromolecules were stimuli-responsive and dynamic in water. Later on, Sawamoto

et al. introduced a new class of amphiphilic random copolymers with an hydrophobic

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Introduction

10 self-assembly behavior due to the formation of intermolecular multi-chain aggregates and intramolecular single-chain unimer micelles[42]_.

Figure 1-6: Self-assemblies of PEGMA/FMA copolymers in water solution. Adapted from ref[42]

Random copolymers of PEGMA and 2-(perfluorohexyl)ethyl acrylate have been studied via fluorescence emission spectroscopy, adding to the system a fluorescent rotor comonomer capable of giving different responses when confined within a unimer micelle rather than free in solution[43]_{. These analyses confirmed the formation}

of unimer micellar structures of the amphiphilic random copolymers in which the hydrophobic fluorescent probe was trapped within the hydrophobic core. Recently, poly(siloxane) methacrylates were incorporated into amphiphilic copolymers as hydrophobic comonomers to form unimer micelles[44]_{. A different approach is to}

create an amphiphilic polymer from a monomer that consists of both hydrophobic and hydrophilic components. To this aim, an aromatic fluorinated monomer, pentafluorostyrene, can be easily modified by nucleophilic substitution preferentially to the para position with a poly(ethyleneglycol) chain to obtain amphiphilic properties. This kind of amphiphilic monomer was used as a comonomer for the preparation of random and block copolymers[45-47]_{. In this way, it is possible to insert}

a wide diversity of functional groups on the FS moiety that can adjust the materials properties of the polymers therefrom according to the final application.

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11 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 structures of SCNPs (Figure 1-7).

Figure 1-7: Structure of sparse (left) and globular (right) SCNPs[48]

PEG-based amphiphilic polymers often show a lower critical solution temperature-(LCST) behavior in water solution. At temperatures above the LCST of the polymers the unimers micelle in water collapse into macro-aggregates due to intermolecular interactions.

1.4 Thermoresponsiveness of amphiphilic

polymers

Thermoresponsive polymers are commonly defined as a class of compounds that exhibit a drastic change in solubility, in a given solvent, as a function of temperature. In particular, such macromolecules have a miscibility gap in their temperature-composition diagram (Figure 1-8).

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Introduction

12

Figure 1-8: Phase diagrams for a binary polymer-solvent mixture with LCST (a) and UCST (b) behavior[49]

Thermoresponsive polymers have a critical solution temperature. Such temperature is that at which the phase of polymer/solvent mixture undergoes a discontinuous change[47]_{. If the decrease in the solubility of the polymer is due to the increase in}

temperature, the system exhibits lower critical solution temperature (LCST) behavior. On the other hand, if phase separation occurs with a decrease in temperature, the polymer has an upper critical solution temperature (UCST). In the literature, many studies are reported on poly(N-isopropylacrylamide) (PNIPAM) with LCST ~ 32 °C (Figure 1-9), which makes this polymer potentially useful for biological applications[47]_{. LCST of PNIPAM can be regulated by copolymerization with}

hydrophilic or hydrophobic comonomers making the total hydrophilicity of the system higher or lower, respectively. This easy LCST regulation can be carried out for most of the thermoresponsive polymers and thus these systems are used in different application fields. Other thermoresponsive polymers are poly(N,N-diethylacrylamide) (PDEAM) with LCST ~ 25-32 °C, poly(vinyl methyl ether) (PVME) with LCST ~ 34 °C, poly(N-vinylcaprolactam) (PVCL) with LCST ~ 35°C, poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) with LCST ~ 50 °C,

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13 poly(2-ethyl-2-oxazoline) (PEOZ) with LCST ~ 62 °C and poly(ethylene glycol) methacrylate (PEGMA) with LCST ~ 65 °C[50]_.

Figure 1-9: Thermoresponsive behavior of PNIPAM. Adapter from ref[51]

1.5 Properties of oxyethylene polymers

Polyoxyethylene chains that are hydroxyl-terminated, HO(CH2CH2O)nH or

HO(CH2CH2O)nR, are normally referred to as PEG, while those with alkoxy

(commonly methoxy) terminal groups, RO(CH2CH2O)nR, are identified as

poly(ethyleneoxide) (PEO). The sequencing of ethylene groups connected by ether C-O bonds 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. PEG has a relatively high surface energy (43 mJ/m2_{) but low interfacial energy with water}

(5 mJ/m2₎[52]_{. PEG is soluble in water and highly hygroscopic due to its capability of}

forming hydrogen bonding with water. Jeon and collaborators[53]_{studied the causes}

that determine the exceptional resistance to adsorption of proteins of PEGylated surfaces. Approach of the protein to the substrate surface causes a compression of the hydrated chains of PEG forming an overall electrostatic force. Another important feature of PEG is being biologically inert and safe, leading it to wide use in the medical field.

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Introduction

14

1.6 Properties of fluorinated polymers

Fluorinated polymers have distinctive properties such as high stability, thermal and chemical resistance and low adhesion due to the nature of the high-energy covalent C-F bond. Despite the excellent chemical-physical properties, the use of fluorinated compounds has been limited by the high cost and poor solubility in common solvents, which causes great difficult in processing.

In a matrix, the fluorinated component allows for segregation and surface migration of the polymer chain. Migration to the surface occurs thanks to the thermodynamic gain following the decrease in the surface energy of the material. The low surface tension is due to the small size of the fluorine atoms, which avoid possible steric stresses of the structure. This causes the fluoropolymers to have a low tendency of the surface to absorb water.

Among the fluorinated polymers, the electronics industry has shown interest in those polymers capable of forming thin films to be used in waveguides, such as poly(pentafluorostyrene) (PFS). In the literature, studies on conventional multi-chain amphiphilic micelles of block copolymers of PFS and PEGMA are reported[54, 55]_.

These systems self-assemble in water forming brush aggregates (diameter ~ 20-60 nm) with the hydrophobic core of PFS block and the shell forming hydrophilic PEGMA chains. The self-assembly of structurally related random copolymers was studied and was found to form small micelles, in the 10-20 nm diameter range, and to also exhibit a LCST[56]_.

1.7 Properties of siloxane polymers

Polysiloxanes, or silicones, are a class of polymers formed by Si-O bonds and organic groups (e.g. methyl groups), which can connect one linear chain backbone or two or more cross-linked chains. By varying the siloxane chain length, side groups and

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15 crosslinking, silicones with properties ranging from liquids to hard plastics can be prepared[57]_{. The most common polysiloxane is poly(dimethyl siloxane) (PDMS).}

These polymers have excellent properties such as biocompatibility, chemical and thermal stability, hydrophobicity and low surface tension. The silicone surface is able to inhibit the obstruction of the blood for many hours and accordingly such materials are used for the manufacture of needles, syringes and other systems for blood collection. Silicone materials may be components for heart valves, kidney dialyzers and heart by-passes due to their blood-compatible behavior.

Silicone coatings have a higher surface energy than fluorinated ones. Moreover, if these coatings are relatively thin, they present poorer antifouling properties than fluoropolymers do[58]_{. To improve the adhesion resistance of silicone-based coatings,}

it is possible to introduce other components such as amphiphilic polymers, zwitterions or quaternary ammonium salts (QAS). Olsen et al.[59] prepa red PDMS-PEG coatings that displayed good antifouling performance for seawater immersion. Galli et al.[60, 61]

investigated the antifouling properties in several cases of PDMS incorporated with amphiphilic polymers.

1.8 Properties of zwitterionic polymers

Zwitterionic polymers represent an innovative class of organic compounds[62]_{. The}

peculiarity of these polymers is the ability to preserve a total zero charge, thanks to the presence of an equal number of positive and negative charges, in a large pH range. Polyzwitterions can be classified as a subclass of polyampholytes. The latter behave mainly as polyanionic or polycationic species, while zwitterionic polymers show a profile more similar to polar non-ionic polymers (figure 1-10).

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Introduction

16

Figure 1-10: Structures of a polyampholyte (left) and a polyzwitterion (rigt)[62]

Zwitterionic polymers are generally formed by five common zwitterionic moieties[63]_:

carboxybetaine (CB), sulfobetaine (SB), phosphatidylcholine (PC), cysteine (Cys) and imidazolium sulfonate (VBIPS) (Figure 1-11).

Figure 1-11: Representative zwitterionic moieties[63]

All zwitterionic polymers show strong hydration in aquoeus solutions by electrostatic interactions. On the other hand, in zwitterionic polymers the different zwitterionic fractions and the distribution of the charges on the macromolecular chains influence the hydration free energy and the hydrated structure[64]_{. Another parameter to be taken}

into account for the design of a zwitterionic polymer is the carbon spacer length (CSL), defined as the number of CH2 methylene groups between the anionic and

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17 hydrophilic/hydrophobic ratio and flexibility of the polymer backbone chain. Molecular dynamics simulations by Shao et al.[65]_{showed the change in the hydration}

state of carboxybetaine as a function of CSL (in the range 0-4). In particular, they found that, on increasing the CSL, the water molecules around the zwitterionic polymer and the residence time increased, while the hydration free energy decreased. Such different behaviors of the structure at the nanoscale could explain the different antifouling behaviors on the macroscale.

Much interest has focused on the preparation of polyzwitterions capable to mimic cell membranes containing phospholipid structures. In this context, Ishihara and co-worker[66]_{designed the synthesis of 2-methacryloyloxyethyl phosphorylcholine}

(MPC) polyzwitterion with a structure similar to the polar group of phospholipids in order to study its resistance to proteins. Later, other zwitterionic polymers based on sulfobetaine or carboxybetaine were prepared and tested to form antifouling materials against microorganism such as Candida Albicans.[67, 68]_.

Another interesting feature of the zwitterionic polymers is that of showing an “anti-polyelectrolyte effect”[69]_{(Figure 1-12) In fact, the polyzwitterion chains, unlike those}

of polyelectrolytes, are more soluble in saline water than in pure water. This happens because the presence of salts shields the inter- and intra-chain electrostatic interactions, breaking the ion pairs and causing the elongation and dissociation of the polymer chains. Exploiting the anti-polyelectrolyte effect of zwitterionic polymers, Hong et al.[70] _{designed salt-sensitive polyVBIPS brushes. These zwitterionic}

materials were tested as reversible systems for protein capture/release from blood plasma in controllable manner.

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Introduction

18

Figure 1-12: Anti-polyelectrolyte effect of zwitterionic polymers[63]

1.9 Applications

In recent decades, polymer micelles have been studied as drug carriers for cancer chemotherapy due to their very well characteristics such as biodegradability and low toxicity[71]_{. Unfortunately, such systems still have several disadvantages such as the}

low quantity and stability of the loaded drug and a lack of drug release triggered by sensitive stimuli in particular micro-environmental conditions. To overcome these issues, another promising approach has been developed with well-controlled single-chain polymeric nanoparticles (SCNPs). These systems have interesting physical properties such as low viscosity, low hydrodynamic volume, controlled affinity and self-assembly ability[19]_{. Preliminary studies suggest that SCNPs may offer better}

stability of the loaded micelles in order to prevent premature drug release, and a more effective ability to trigger drug release into the tumor micro-environment, which generally exhibits slightly acidic conditions and a temperature above normal. Obviously, in order to exploit the thermoresponsiveness of SCNPs for drug delivery systems, the LCST of these polymers must be close to that of the human body. Cheng

et al.[72] showed that SCNPs constituted by a copolymer PEGMA containing uracil-diamino-pyridine groups, capable of crosslinking via hydrogen bonds, can stably encapsulate a hydrophilic anticancer drug, 5-fluoroacil, with an effective release triggered simultaneously by temperature near or above LCST (44 °C) and an acidic condition (pH = 4). Recently, Sawamoto and co-workers reported the effective

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19 encapsulation of a fluorinated agrochemical, such as novaluron, within self-assembled nanostructures of fluorinated amphiphilic random copolymers[73]_.

Another potential application of single-chain folded nanoparticles is in the field of catalysis. For example, PMMA-based SCNPs synthesized by Zhao and co-workers[74]

were described as nanoreactors for the synthesis in situ of gold nanoparticles. Meijer and collaborators prepared a water-soluble amphiphilic terpolymer (Ru-PEGMA/BTMA/SDP) capable of catalysing the reduction of cyclohexanone to cyclohexanol[40]_{(Figure 1-13).}

Figure 1-13: Supramolecular single-chain folding of Ru-PEGMA/BTMA/SDP terpolymers in water. Adapted from ref[40]

The synthesis of oxidase enzymes-mimicking SCNPs is reported[75]_{. These}

metal-folded SCNPs including complexed Cu(II) ions demonstrated high catalytic selectivity during the oxidative coupling of acetylene-terminated chemical substrates. In nanomedicine SCNPs could also be used as image contrast agents for magnetic resonance, fluorescence imaging and tomography[18]_{. Such application is possible}

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Introduction

20 through photochemical design of functional SCNPs or intrisic contrast, for example, of perfluorinated containing compounds in 19_{F magnetic resonance.}

On the other hand, amphiphilic polymers can also effect self-assembly behaviors in the bulk phase. Examples of self-assembled nanostructures were created at the outer surface of thin films in order to develop anti(bio)fouling surfaces to combat ,adhesion of proteins, cells or organisms[76]_{. Thus, films of amphiphilic (co)polymers appear to}

be optimal candidates for application in the marine and maritime fields, where natural and artificial surfaces are inevitably colonized from adhered microfoulants and macrofoulants which causes detrimental effects on health, environment and costs[76]_.

1.10 Biofouling

The word biofouling identifies the accumulation and uncontrolled growth of biomolecules, micro- and macro-organisms on wet surfaces. Therefore, this undesired phenomenon constitutes a general major problem for devices operating at interfaces, such as those in contact with sea water, e.g. boats, pipes, off-shore structures, or in contact with biological fluids, e.g. catheters, stents, contact lenses, prostheses (Figure 1-14).

Figure 1-14: Examples of areas of the human body susceptible to infectious biofilms (left) and marine biofouling (right). Adapter from ref [77]

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21 The undesired colonization of organisms on the hulls leads to a reduction in performance and, therefore, higher fuel consumption. As a result, total costs and emission of noxious substance into the environment increase. Moreover, dirty ships can transport alien invasive species in non-native ecosystems, causing very serious damage to biodiversity.

Settlement of biomolecules can be influenced by the chemical composition of the substrate on which adhere. Surface energy is one of the most important chemical-physical parameters that effect the formation of the biofilm. Furthermore, the roughness and porosity of the surface play a role on the settlement; in fact, the irregularities of the surface result in an increase in the colonizable surface.

Hydrophilic surfaces are more resistant to non-specific adsorption of proteins than hydrophobic substrates. This is because when approaching a hydrophobic surface, the proteins in their native state undergo a series of conformational changes with a first reversible adsorption, up to irreversible settlement through subsequent steps. To obtain the complete formation of the biofilm, there must be enough surface area for anchoring to the substrate. In this context, a potential solution to the problem of marine fouling is the design of non-toxic coatings where the molecular forces between the microorganisms and the surface are minimized in order to obtain fouling release. This type of antifouling coatings are based on the spontaneous formation of self-assembled nanostructures under specific conditions. In recent years, different approaches have been investigated such as biopolymers incorporating bioactive molecules and enzymes[78]_{, self-assembled hydrophobic or hydrophilic monolayers}[79]_{, hydrophobic}

polymeric networks[80]_, _quaternary _ammonium _compounds[81]_, _hybrid

polysiloxanes[82]_{, zwitterionic polymers}[83]_.

Another very promising recent strategy implies the use of amphiphilic polymers. The potential of these macromolecules is to show an “ambiguous” chemically

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Introduction

22 heterogeneous surface in which the coexistence of hydrophobic and hydrophilic domains can confuse organisms during their settlement and adhesion phases. In the biomedical field, the formation of biofilm on medical devices leads to an increase in the proliferation of bacteria and so the patient can easily contract infections and inflammations with the most common fungal infections due to the yeast Candida

albicans. Several studies showed that C. albicans adheres more rapidly to

hydrophobic and non-polar surfaces, such as Teflon, than to hydrophilic materials, such as glass[84]_{. For this reason, attempts have been made to modify prosthetic}

materials to make them more hydrophilic and less prone to biofouling. For example, Lazarin et al.[85]_{foun d that denture-base acrylic resins coated with sulfobetaine-based}

zwitterionic polymers presented lower adesion of C. albicans than untreated acrylic resins. Zwitterionic materials can be considered for effective hydrophilic antifouling coatings thanks to their ability to strongly bind water molecules through electrostatic interactions, so as to make the settlement of biomolecules thermodynamically unfavorable.

1.11 Conventional radical polymerization

Conventional radical polymerization is a chain reaction, where the vinyl functional groups of a monomer react by addition with radical species. When mixtures of two (or more) suitable such monomers are fed for the reaction, copolymers are normally formed that incorporate counits from both reacted monomers. The composition and distribution of the comonomer units of the polymers synthesized by this type of polymerization are controlled via kinetics aspects. The lifetime of an individual radical is very short (< 1 s) and only in that moment the growth of the chains is possible. In a radical polymerization the formation of primary radicals by homolytic split of bond is necessary. Azo compounds, e.g. azo-bis(isobutyronitrile) (AIBN) and peroxides, e.g. dibenzoyl peroxide (BPO) (Figure 1-15) can be used as radical

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23 initiators: the presence of highly unstable bond causes the formation of radicals with the increase in temperature (thermal activation) or under the incidence of UV rays on the molecule (photochemical activation) which will react with the monomers.

Figure 1-15: Chemical structure of AIBN (left) and BPO (right) thermal initiators

The half-life time, correlated to the reactivity of initiator, strongly influences the speed of the overall reaction, the number of growing macroradicals and therefore the length of the polymers obtained. High values of the half-life time, given by a poor reactivity of the initiator or low reaction temperature, can favor the formation of high molecular weight polymer chains.

In this kind polymerization, three fundamental steps of the process are generally acknowledged: - chain initiation - 𝑀 + 𝐼 · → 𝑃1· - chain propagation 𝑃1· + 𝑛𝑀 → 𝑃𝑛· - chain termination 𝑃𝑛· + 𝑃𝑚·→ 𝑃 𝑛+𝑚 𝑃𝑛· + 𝑃𝑚·→ 𝑃 𝑛 + 𝑃𝑚

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Introduction

24 The conventional radical polymerization is tolerant to protic solvents and impurities, such as oxygen, and therefore can be carried out in water and other polar solvents even with monomers not severely dried. These characteristics place this method among the most important industrial polymerization techniques with about 40-45% of commercial synthetic polymers prepared with conventional radical polymerization[86]_.

1.12 Reversible-deactivation radical

polymerization

Many applications benefit from the development of functional polymers with well-defined structures. For this reason, several studies have focused on the design of controlled synthesis methods through the reversible deactivation of chain transporters. The first approach to controlled radical polymerization was introduced in 1982 by Otsu[87]_{through the use of the so-called “iniferter”}

(initiator-transfer-agent-terminator) as photochemical initiators. Later, in the 1990s, new “living” polymerization processes were developed[88]_{. These methods were defined “living”}

since once the monomer in feed is exhausted, the macroradicals remain active in a quiescent phase, waiting for another monomer or a terminator. In 2010 IUPAC[89]

proposed the term reversible-deactivation radical polymerization (RDRP) for such processes, commonly referred to as controlled radical polymerizations or living radical polymerizations. A number of different techniques RDRP have been developed, notably the nitroxide-mediated polymerization (NMP), reversible addition-fragment chain transfer (RAFT) polymerization, and atom-transfer radical polymerization (ATRP). These various methods follow similar mechanisms (Figure 1-16). Most chains are in deactivated, dormant form and only a small fraction of active chains is able to propagate the radical reaction. This decreases all the

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25 termination phenomena that occur in a conventional radical polymerization process and allows the polymer chains to grow at the same speed.

Figure 1-16: General mechanism of controlled radical polymerization

The first industrially applied RDRP reaction was the iodine-transfer polymerization for preparation of fluorinated thermoplastic elastomers[90]_.

Polymers synthesized via RDRP are expected to find application in various fields such as aerospace, automotive, biomedical, cosmetics, coatings, electronics and nanotechnology. Recently, scientific interest has focused on still improving these polymerization techniques to solve some disadvantages associated with them, such as residual catalyst metals in ATRP products or nature of terminal groups in RAFT. Furthermore, the control agents must be cost-effective and environmentally sound.

1.12.1 Atom transfer radical polymerization (ATRP)

In ATRP, the growing chain is reversibly activated by the metal catalyst. For this kind of polymerization monomers, initiator, metal catalytic complex and ligand are necessary. As shown in Figure 1-17, the control on polymeric structure occurs by the balance between the activation reaction of dormant species Pn-X and the deactivation

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Introduction

26 suitable ligand (L) catalyzes the activation/deactivation reactions. This metal must be able to reversibly pass from a lower to a higher oxidation state (Mtn_/Mtn+1_{) by means}

of a redox reaction between metal halide and alkyl halide.

Figure 1-17: General ATRP mechanism. Adapted from ref[91]

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 initiator is a molecule that contains a mobile halide (X), activated by a substituent such as α-carbonyl, phenyl, or vinyl. The action of the initiator determines the number of growing polymeric chains and therefore the final molecular weight of the polymer. The choice of the ATRP initiator is influenced by two important parameters. First, the initial phase of the process must be fast with respect to propagation. Second, the propagation of secondary reactions must be minimized. The activity of the initiator depends on the degree of substitution (primary < secondary < tertiary), the transfer group (Cl < Br < I) and the stabilization of the radical (-Ph, -COOR << -CN). Dentate ligand regulates redox potential of the equilibrium reaction and promotes the solubility of the metal halide in the chosen solvent. Moreover, the reactivity of the catalyst is also highly influenced by steric and electronic properties of the ligand. Although secondary reactions are minimized, chain termination always occurs to some degree. However, the termination of radical propagation in the ATRP initial phase can be advantageous as the presence of a slight excess of deactivating species

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27 provides further control over the molecular weight distribution by shifting the equilibrium toward the dormant species. This phenomenon is known as the persistent radical effect (PRE).

Many monomers have been successfully polymerized, such as acrylates and methacrylates, styrenes, acrylamides and acrylonitrile. Depending on the monomer, the reaction conditions, catalyst, initiator and ligand system must be optimized to obtain a good control over the polymerization.

The main disadvantages of this kind of controlled radical polymerization for an industrial scale-up are high sensitivity to trace oxygen impurities and the relatively large amount of transition metal catalyst that must be used, resulting in increased costs of the final steps of polymer purification. Over the years, several studies have been carried out to reduce the catalyst loading to parts per million levels in order to effect more environmentally friendly syntheses[92]_.

1.12.2 Activators regenerated by electron transfer

(ARGET)

Activators ReGenated by Electron Transfer (ARGET) is a variant of ATRP process, commonly named ARGET-ATRP, for it uses much lower concentrations of catalyst to obtain a “green” procedure. As shown in Figure 1-18, in this polymerization Cu(I) complexes are regenerated from stable oxidized Cu(II) species by the action of a reducing agent (tin(II) 2-ethylhexanoate, glucose, ascorbic acid, silver metal, hydrazine) inside the reaction system. The excess of deactivating Cu(II) accumulated due to the inevitable termination reactions in ATRP determines serious consequences for the polymerization. In ARGET-ATRP, this problem is solved as the excess of the deactivating agent is continuously reduced to the activating species. In addition, the use of the reducing agent allows a greater tolerance to the presence oxygen in the

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Introduction

28 environment to be gained, rendering the experimental conditions less demanding. In fact, is possible to introduce the catalyst in its oxidized form, i.e. as Cu(II), and reduce it in situ, unlike in ATRP.

Figure 1-18: General ARGET-ATRP mechanism[91]

The literature reports controlled syntheses of a wide range of polymers using ARGET-ATRP such as polystyrene (PS), poly(methyl methacrylate) (PMMA), poly(n-butyl acrylate) (PnBA) and poly(2-hydroxyethyl methacrylate) (PHEMA). In some cases, ARGET-ATRP allows the synthesis of polymers that cannot be polymerization by conventional ATRP, such as high molecular weight acrylonitrile copolymers. Moreover, the development of a novel ARGET-ATRP without the addition of an external reducing agent is considered very interesting, for instance when using systems with a ligand that has tertiary amine group or monomers, such as (2-dimethylaminoethyl methacrylate) (DMAEMA), which can act as intrinsic reducing agents[93]_.

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29

1.12.3 Reversible addition-fragmentation

chain-transfer (RAFT)

The first report on the RAFT method dates back to 1998 by Rizzardo and collaborators who realized this new controlled polymerization technique in the absence of heavy metals[94]_.

An important difference compared to other controlled radical polymerizations is the use of a conventional radical initiator, e.g. AIBN. In this way, the number of chains that irreversibly terminate is directly proportional to the concentration of the initiator and therefore it is easier to modulate the polymerization speed and the numerical fraction of living chains. RAFT polymerization can be considered a special case of degenerative transfer (DT), based on the reversible action of a chain transfer agent (CTA). This is generally known as the RAFT agent, and is depicted in Figure 1-19, where X is usually sulfur, C=X is a reactive double bond, R is a leaving group and Z can take on different structures.

Figure 1-19: General structure of a RAFT agent

The most common RAFT agents are aromatic and aliphatic dithioesters, trithiocarbonates and dithiocarbamates. These compounds are widely used, thanks to their low cost and the absence of potentially unwanted terminal groups within the product.

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Introduction

30 RAFT polymerization, as in a conventional radical, is initiated by an initiator usually of the thermal type (azo-compounds or peroxides), which forms primary radicals due to the homolytic breaking of a bond (Figure 1-20). This leads to the formation of an oligoradical (Pn·), which will react with the RAFT agent to form a radical intermediate

releasing the R group in radical state. When all the molecules of the RAFT agent are converted into dormant species, a balance of activation/deactivation of the growing polymer chains is established by a mechanism defined as “addition-fragmentation”. During this phase, a radical intermediate is present, called degenerate since it carries two chains linked to the two sulfur atoms, which differ only in the degree of polymerization (Figure 1-20). Polymerization stops following the exhaustion of monomers, except for possible unwanted termination reactions that may occur leading to the formation of dead polymer chains (5-10 wt% of the final product).

Figure 1-20: General mechanism of RAFT[95]

In this process the growth of the chains occurs by the macroradicals when they are separated from the RAFT agent (active chains), whereas they are inactive (dormant

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31 form) when the polymer chain is linked to the CTA. In order for a RAFT process to be efficient, there must be a quick balance in the transition from dormant to active chains so that all chains grow equally, ensuring optimal control over the degree of polymerization and the dispersion of molecular weight.

An important feature of RAFT polymerization is the ability to be applied to a wide variety of functional monomers. The high versatility of this kind of synthesis is related to the reactivity of the CTA. Generally, a RAFT polymerization will be successful if the C=S double bond is more reactive towards the radical species than the monomer. This can be obtained by a good choice of RAFT agent Z and R groups. The Z group is mainly responsible for the reactivity of the C=S double bond towards radicals and the stability of the intermediate radical. Instead, the R group must be a good leaving group forming a radical that is enough stable but also reactive towards the monomer. Generally, best R groups are those whose radicals have a behavior similar to the monomeric or thermal initiator radicals.

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Introduction

32

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