Designing Polymers forBiological Interfaces - From

Antifouling to Drug Delivery

P O N T U S L U N D B E R G

Doctoral Thesis in Polymer TechnologyStockholm, Sweden 2010

DESIGNING POLYMERS FOR BIOLOGICAL INTERFACES - FROM

ANTIFOULING TO DRUG DELIVERY

Pontus Lundberg

AKADEMISK AVHANDLING

som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknisk doktorsexamen fredagen den 10 december 2010, kl 10:00 i sal F3, Lindstedtsvägen 26, KTH, Stock-holm. Avhandlingen försvaras på engelska. Opponent: Professor Scott M. Grayson från Tulane University, USA

Copyright © 2010 Pontus Lundberg All rights reserved

Paper I © 2010 American Chemical SocietyPaper III © 2010 The Royal Society of Chemistry

TRITA-CHE-Report 2010:50ISSN 1654-1081ISBN 978-91-7415-802-1

ABSTRACT

Unspecific interactions, at the interface between a synthetic material and an aqueous biological environment, leading to irreversible protein adsorption can cause to undesired consequences. These include fouling of a boat hull or a triggered immune response. Thus, stealthy materials are a topic that has generated a great deal of interest in the scientific community. This work deals with the design of networks, nanoparticles, and surfaces containing poly(ethylene glycol) (PEG), known for its resistance to protein adsorption and non-toxic nature. Initially, PEG-based networks, hydrogels, were synthesized using photoinduced thiol-ene chemistry in order to afford coatings targeted for marine antifouling applications. By varying the length of the PEG chain, curing chemistry, cross-linker as well as hydrolytical stability, a library of hydrogel coatings was produced. The coatings were subsequently characterized with respect to curing efficiency, thermal and mechanical properties, and aqueous stability. Furthermore, the antifouling properties of coatings were evaluated using in vitro tests with proteins, marine bacteria, and diatoms. As a final test the coatings were evaluated in a four month field test. It was found that coatings comprising longer PEG chains displayed enhanced antifouling performance, compared to shorter PEGs. In addition, the choice of cross-linker, curing chemistry, and hydrolytical stability also affected the properties to a great extent. This thesis further deals with the design of amphiphilic linear dendritic hybrids, with PEG as the hydrophilic block. Using non-toxic 2,2-bis(methylol) propionic acid (bis-MPA) based dendrons, bearing click functional cores (alkyne or allyl) and peripheral hydroxyl groups, as macrointitiators for ring-opening polymerization of ε-caprolactone, a library of star branched materials was afforded. As a final step, click functional (azide or thiol) PEGs were attached using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or thiol-ene click chemistry. The size of the dendrons was varied from generation 0-4, along with variation of both poly(ε-caprolactone) (PCL) length and PEG length. The materials were designed in order to allow a study of the impact of the dendron generation. Finally, the hybrid materials were used for the preparation of micelles, as well as for the formation of honeycomb membranes. The micelles critical micelle concentration, size and drug loading capacity were shown to be highly dependent on the generation of the dendron. The generation of the dendron also had a profound effect on the ability of the hybrid materials to form ordered honeycomb membranes, and hybrid materials of the 3rd generation yielded the most highly ordered membranes.

SAMMANFATTNING

Ickespecifika interaktioner vid gränsytan, mellan ett syntetiskt material och en vattenbaserad biologisk miljö, kan leda till irreversibel adsorption av proteiner. Detta kan i sin tur leda till oönskade följdeffekter, såsom beväxning på båtskrov eller trigga en immunologisk reaktion. För att motverka dessa effekter har forskare utvecklat så kallade smygmaterial. Denna avhandling behandlar design av nätverk, nanopartiklar och ytor innehållande poly(etylenglykol) (PEG), som är känt för sina smygegenskaper och för att vara icke-toxiskt. Initialt behandlar avhandlingen PEG-baserade nätverk, hydrogeler, syntetiserade med fotoinitierad tiol-enekemi, för användning som beväxningsavvisande beläggningar för marina applikationer. Genom att variera olika parametrar, såsom längden på PEG-kedjan, härdningskemin, tvärbindaren samt den hydrolytiska stabiliteten, byggdes ett bibliotek av hydrogelbeläggningar upp. Hydrogelbeläggningarna karaktäriserades sedan med avseende på härdningseffektivitet, termiska och mekaniska egenskaper, samt hydrolytisk stabilitet. Vidare studerades beläggningarnas avvisande förmåga mot proteiner, bakterier samt kiselalger. Slutligen studerades ytbeläggningarna i ett fyra månader långt fälttest. Av testerna framgick att längre PEG-kedjor gav beläggningar med bättre avvisande förmåga. Dessutom framgick att valet av tvärbindare, härdningskemi samt hydrolytisk stabilitet var av betydelse för beläggningarnas effektivitet. Denna avhandling behandlar vidare design av amfifila linjära dendritiska hybridmaterial, med PEG som den hydrofila delen. Genom att använda icke-toxiska 2,2-bis(metylol)propionsyrabaserade dendroner, med en klickfunktionalitet i kärnan (alkyne eller allyl) och perifera hydroxylgrupper, som makroinitiatorer för ringöppningspolymerisation av ε-kaprolakton byggdes ett bibliotek av material upp. För att göra materialen amfifila, kopplades klickfunktionella PEG-kedjor (azid eller tiol) till kärnan med koppar(I)-katalyserad azid-alkyn cykloadditionskemi alternativt tiol-enekemi. Storleken på dendronerna varierades från generation 0-4, dessutom varierades längden på både poly(ε-kaprolakton)- och PEG-kedjorna. Materialen designades så att inverkan av dendrongenerationen kunde studeras. Slutligen användes dessa hybridmaterial för att framställa miceller samt isoporösa filmer. Micellernas kritiska micellbildningskoncentration, storlek samt förmåga att laddas med läkemedel visade sig vara mycket beroende av dendrongenerationen. Dendrongenerationen visade sig vidare även ha stor inverkan i hybridmaterialens förmåga att självorganisera sig till en isoporös struktur och material av tredje generationen gav de mest välordnade filmerna.

Till Pappa

LIST OF PAPERS

This thesis is a summary of the following papers:

I. “Poly(ethylene glycol)-based thiol-ene hydrogel coatings – curing chemistry, aqueous stability, and potential marine antifouling applications” P. Lundberg, A. Bruin, J. W. Klijnstra, A. M. Nyström, M. Johansson, M. Malkoch and A. Hult, ACS Applied Materials & Interfaces, 2010, 2, 903-912.

II. “Poly(ethylene glycol)-based thiol-ene hydrogels evaluated for their potential as antifouling coatings: Field studies and mechanical properties” P. Lundberg, B. Daehne, O. Andrén, R. Laurin, B. Watermann, C. Lundin, M. K. G. Johansson, M. Malkoch, and A. Hult, Manuscript

III. “Linear dendritic polymeric amphiphilies with intrinsic biocompatibility: synthesis and characterization to fabrication of micelles and honeycomb membranes” P. Lundberg, M. V. Walter, M. I. Montañez, D. Hult, A. Hult, A. Nyström and M. Malkoch, Polymer Chemistry, DOI: 10.1039/c0py00258e

IV. “Honeycomb films from amphiphilic linear-dendritic-linear hybrids: effect of branching and of the block length”, M. V. Walter, P. Lundberg D. Hult, A. Hult and M. Malkoch, Manuscript

My contribution to the appended papers:

I. All synthetic work, all chemical analysis and most of the preparation of the manuscript.II. All of the synthesis, most of the coating preparation, most of the mechanical analysis and most of the preparation of the manuscript.III. All of the synthetic work, most of the analysis and most of the preparation of the manuscript.IV. Most synthetic work, parts of the analysis and some of the preparation of the manuscript.

Scientific contributions not included in this thesis:

V. ”Controlled design of amphiphilic block-copolymers using ring- opening polymerization and click chemistry”, P. Lundberg, P. Antoni, L. Fogelström, M. Malkoch, A. Hult, Polymer Preprints (American Chemical Society, Division of Polymer Chemistry), 2007, 48(2), 407-408.

VI. “Click Assisted One-Pot Multi-Step Reactions in Polymer Science: Accelerated Synthetic Protocols “ P. Lundberg, C. J. Hawker, A. Hult, M. Malkoch, Macromolecular Rapid Communications, 2008, 29, 998- 1015.

VII. “One-pot dendritic growth and post-functionalization of multifunctional dendrimers: Synthesis and application” P. Antoni, D. Nyström, P. Lundberg, A. Hult and M. Malkoch, Manuscript.

VII. “Thiol macrodendrons: a new tool for the facile synthesis of dendritic polymers“ M. V. Walter, P. Lundberg, A. Hult and M. Malkoch, Manuscript.

TABLE OF CONTENTS

1. PURPOSE OF THE STUDY ...........................................................................1

2. INTRODUCTION .............................................................................................32.1 Protein adsorption .......................................................................................32.2 Poly(ethylene glycol) ....................................................................................42.3 Chemical toolbox .........................................................................................5

2.3.1 Click chemistry ....................................................................................52.5.1.1 CuAAC........................................................................................62.5.1.2 Thiol-ene chemistry .........................................................................7

2.3.2 Architecturally advanced polymers ..................................................92.3.2.1 Dendritic polymers ..........................................................................92.3.2.2 Linear dendritic hybrid materials ..................................................12

2.3.3 Ring-opening polymerization of cyclic esters .............................132.4 Applications ................................................................................................15

2.4.1 Marine antifouling ............................................................................152.4.1.1 Biofouling .....................................................................................152.4.1.2 Development of antifouling technologies .........................................162.4.1.3 Non-toxic antifouling strategies .....................................................17

2.4.2 Isoporous membranes .....................................................................182.4.3 Drug delivery .....................................................................................19

3. EXPERIMENTAL ...........................................................................................233.1. Materials .....................................................................................................233.2 Instrumentation .........................................................................................243.3 Synthetic, preparative and analytical procedures...................................25

3.3.1 Hydrogel coatings .............................................................................253.3.1.1 Preparation of thiol functional glass surfaces .................................253.3.1.2 General coating procedure for hydrogel coatings H1-H8 on glass slides ..........................................................................................263.3.1.3 Application of primers on PVC panels ........................................263.3.1.4 Application of hydrogel coatings on primed PVC panels ..............263.3.1.5 Swelling and stability studies .......................................................263.3.1.6 Protein adsorption studies ............................................................273.3.1.7 Bioassays .....................................................................................273.3.1.8 Field studies .................................................................................283.3.1.9 Mechanical properties of coatings .................................................28

3.3.2 Linear dendritc hybrids ....................................................................293.3.2.1 Polymerization of ε-CL using dendrons with alkyne or allyl functional cores as initiators .........................................................................293.3.2.2 Synthesis of amphiphilic linear dendritic hybrid block-copolymers using CuAAC chemistry .............................................................293.3.2.3 Synthesis of amphiphilic linear dendritic hybrid block-copolymers using thiol-ene chemistry ...............................................................303.3.2.4 Formation of self-assembled nanoparticels from amphiphilic linear dendritic blockcopolymers ....................................................303.3.2.5 Formation of isoporous films from amphiphilic linear dendritic blockcopolymers .............................................................31

4. RESULTS AND DISCUSSION .....................................................................334.1 Design of hydrogel coatings and their potential as antifouling coatings .......................................................................................................33

4.1.1 Synthesis of monomers and crosslinker .......................................334.1.2 Hydrogel coating preparation, curing and physical properties ..354.1.3 Thermal properties of starting materials and hydrogels ............384.1.4 Swelling and degradation .................................................................414.1.5 Mechanical properties ......................................................................414.1.6 In vitro antifouling studies ................................................................444.1.7 Field tests of hydrogel coatings ......................................................45

4.2 Synthesis of linear dendritc hybrids and their potential applications 464.2.1 Synthesis of linear dendritic hybrid block-copolymers using ROP and click chemistry ................................................................474.2.2 Applications of the amphiphilic linear dendritic hybrids ...........51

4.2.2.1 Formation of nanosized micelles ...................................................514.2.2.2 Formation of ordered honeycomb membranes.................................54

5 CONCLUSIONS ...............................................................................................57

6 FUTURE WORK ...............................................................................................59

7 ACKNOWLEDGEMENTS ............................................................................61

8 REFERENCES ..................................................................................................63

1

1 PURPOSE OF THE STUDY

1. PURPOSE OF THE STUDY

At the interface of a synthetic material and an aqueous biological environment, such as the ocean or the human body, proteins and other biomacromolecules are always present. The proteins are readily adsorbed to any surface and in many cases this adsorption is irreversible. This process can lead to several undesired consequences, including heavy fouling of a boat hull or a triggered immune response. In order to avoid this process scientists have researched the topic of stealthy materials. One polymer that has shown to possess these stealthy properties, regarding protein adsorption, is poly(ethylene glycol) (PEG). PEG is also known to be non-toxic, and is available at a low cost. This thesis focuses on the design of networks, surfaces and nanoparticles containing PEG for applications such as marine antifouling, isoporous membranes, and drug delivery.

In order to further explore the potential of using PEG as a stealthy material in marine antifouling applications, hydrogel coatings, with PEG as the major component, were prepared and evaluated. Thus, a library of hydrogel materials was prepared using efficient thiol-ene chemistry. The effect of different parameters, such as PEG length, curing chemistry and chemical stability, has been investigated, as well as the performance of the coatings in both in vitro studies as well as in a field study.

Other areas where protein adsorption could have negative impact are within the biomedical field. Amphiphilic materials are, with its ambivalent nature, of great interest for potential applications including drug delivery systems, sensors, and miniature reactors. Thus, amphiphilic materials having a stealthy hydrophilic part could be of great importance. Architecture is a powerful tool to tailor the properties of amphiphilc materials. Hence, we targeted the investigation of amphiphilic linear dendritic hybrid materials and the effect of architecture. Using 2,2-bis(methylol) propionic acid (bis-MPA) based dendrons, poly(ε-caprolactone) (PCL) as the hydrophobic block and PEG as the hydrophilic block a library of amphiphilic linear dendritic hybrids were synthesized. Some potential applications, including isoporous membranes, with potential use as e.g. miniature reactors or sensors, and drug delivery systems, were investigated.

3

2 INTRODUCTION

2. INTRODUCTION

In biological environments, such as the ocean or the blood stream, the chemistry is very complex and at any given moment there are H2O, O2, CO2, small organic molecules, salts and minerals, as well as a large number of organic macromolecules present. The latter includes proteins, polysaccharides, proteoglycans, and many others. A surface introduced to such an environment will almost instantaneously be covered with adsorbed macromolecules. This process could be either reversible or irreversible depending on the chemistry of the surface. Irreversible adsorption can, in turn, be a good primer for bacterial growth or trigger an immune response. Hence, this type of adsorption is, in many cases, undesired and to avoid this problem the design of stealthy surfaces and particles is of great interest for a range of applications. To address these challenges, the scientific community has put a lot of effort into the understanding of this process and how adsorption can be avoided. One synthetic polymer capable of producing stealthy surfaces is PEG. In order to elucidate the concerned topics, the following introduction deals with protein adsorption, PEG, the chemical toolbox required for the synthesis as well as the applications such as marine antifouling, isoporous membranes and drug delivery.

2.1 Protein adsorption

In order to avoid irreversible protein adsorption it is crucial to understand underlying mechanisms. Proteins are polymeric amphiphiles capable to adapt to the surrounding chemical environment. In aqueous environments most of the hydrophilic parts of a protein can be found at the protein-water interface, whereas the hydrophobic parts are internalized within the proteins. Upon facing a hydrophobic surface, proteins have high affinity for becoming irreversible adsorbed due to unfolding of the protein, exposing its hydrophobic parts to the surface. The unfolding is accompanied by the release of water from the molecules at the interface leading to a large entropy gain of the system. Charged surfaces can also form strong bonds to proteins due to coulombic interactions even though the charges are highly shielded in marine or physiological conditions. Once a monolayer of proteins is adsorbed to a surface, the adsorbed proteins act as a nonfouling surface with respect to other proteins. Cells; however, can

4

2 INTRODUCTION

continue the fouling process and irreversibly adsorbed proteins are considered to act as a conditioning layer for bacteria. It is also recognized that surfaces containing multiple strong hydrogen bonding groups are susceptible for irreversible protein adsorption. Taking this knowledge into account it can be concluded that a hydrophilic, neutral surface that lacks strong hydrogen bonding groups should be a good start as a nonfouling surface with regards to proteins1,

2.

2.2 Poly(ethylene glycol)

PEG is a semi-crystalline, water soluble neutral polymer. It is also known under the synonyms poly(ethylene oxide) (PEO), polyoxyethylene (POE) and polyoxirane, although PEG is traditionally used for polymers up to 20000 Da and PEO for higher molecular weight polymers. In this thesis PEG will be used to describe all molecular weights. The first reports about PEG dates back all the way to 18593; however, high molecular weight polymers were not commercially available until the late 1950s. PEG can be synthesized from either ethylene glycol (only shorter polymers can be prepared) or more commonly ethylene oxide4 (Figure 2.1).

O O

Ethylene oxide Poly(ethylene glycol) (PEG)

Figure 2.1. Ethylene oxide and its polymer poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO).

PEG is known to be highly protein resistant, which is attributed to its hydrophilicity, neutrality, absence of hydrogen donors and highly dynamic nature. The effect of molecular weight of the PEG has not been fully elucidated as some studies indicate that higher molecular weights are required to afford protein resistance, whereas others have indicated that even short oligomers can afford protein resistance1, 2, 5.

PEG is also known to possess good biocompatibility6, 7. Several studies have shown that PEG is non-toxic and is mainly cleared from the blood by the kidneys. PEGs with molecular weights up to 4000 Da are readily cleared within hours. Higher molecular weight PEGs require longer clearance times but are eventually also cleared. PEGs with molecular weights less than 400 Da are less biocompatible as they may be oxidized in vivo to toxic diacids and hydroxyl acid

5

2 INTRODUCTION

metabolites7. In the natural environment there are several bacteria, both aerobic and anaerobic, that degrade PEG enzymatically, of which some can even use PEG as a carbon source6.

Due to its availability, protein resistance and biocompatibility, PEG is used for a wide range of applications ranging from non-ionic surfactants in detergents and soaps to a range of different biomedical applications. Recently the use of PEG in marine antifouling has also become an area of great interest, vide infra.

2.3 Chemical toolbox

In order to design and produce the structures, networks, surfaces and nanoparticles included in this thesis a range of different synthetic approaches have been used. This chapter deals with the most important reactions and strategies for their preparation.

2.3.1 Click chemistry

The adaptation of synthetic methodologies used in organic chemistry to polymer chemistry is often difficult due to the additional constraints imposed by the macromolecular framework. In organic chemistry, the use of highly specific and efficient reactions can increase the yield of a specific product. However, dealing with polyfunctional macromolecules a highly specific and efficient reaction is of absolute necessity in order to obtain the desired macromolecules8, 9. A class of reactions that could fulfill these requirements is the click reactions. Click chemistry, as introduced in 2001 by Sharpless and coworkers10, stipulate that a click reaction should be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and stereospecific. Furthermore, the reaction should require simple reaction conditions, and readily available starting materials and reagents. The solvent used should be benign or easily removed. These requirements can be fulfilled if the thermodynamic driving force is high enough, usually greater than 20 kcal∙mol-1. Such reactions are said to be “spring-loaded” for a simple trajectory and include: cycloadditions of unsaturated species, especially 1,3-dipolar cycloaddition reactions but also the Diels-Alder family of transformations; nucleophilic substitution chemistry, particularly ring-opening reactions of strained heterocyclic electrophiles; carbonyl chemistry

6

2 INTRODUCTION

of the non-aldol type; additions to carbon-carbon multiple bonds, especially oxidative cases, but also Michael additions of Nu-H reactants. More recently, the orthogonal properties of many click reactions has been appreciated as this allows for more efficient synthetic protocols11, 12. Two types of orthogonal click reactions have received more attention, namely the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and more recently thiol-ene chemistry.

2.5.1.1 CuAACThe CuAAC13, 14 reaction is a modification of the classical Huisgen 1,3

dipolar cycloaddition between azides and alkynes15. The classical Huisgen reaction needs thermal activation and produces a mixture of regioisomers, i.e. 1,4 and 1,5 1,2,3-triazoles (Scheme 2.1), whereas the CuAAC reaction produces the 1,4 regioisomer exclusively. A mechanism for the reaction was proposed in the initial publications by Meldal and coworkers14 and Sharpless and coworkers13 in 2002 but have later been revised and the current accepted mechanism was proposed in its present form by Maarseveen and coworkers16 in 2006 (Scheme 2.2). A range of different Cu(I) sources has been used as catalysts whereof the most common in organic solvents are CuBr and CuI. In aqueous systems the in situ generation of Cu(I) from CuSO4 by sodium ascorbate have been used with great success. Metallic Cu(0) have also been used as well as other metals, such as Ru, Pd2+, Pt2+, and Ni2+. There are also a number of metal-free alternatives especially suited for biological applications17-20. As it is outside the scope of this thesis to elucidate the use of different catalytic systems and the application of click chemistry in macromolecular engineering, the reader is advised to read some of the many reviews on the subject for a complete picture and reference list11, 12, 21-42. The number of reviews and their diversity clearly shows the impact and applicability of the CuAAC reaction in field of polymer science.

R'

N N+

NR

NN

NR'

RN

NN

RR'

Δ ++

Scheme 2.1. The thermally activated Huisgen 1,3 dipolar cycloaddition forming 1,4 and 1,5 1,2,3 triazoles.

7

2 INTRODUCTION

2.5.1.2 Thiol-ene chemistryInitial reports on the reaction of thiols and enes came as early as 190543

and showed that thiols and enes reacted spontaneously or in the presence of an acid. The first reports of polymerizations involving the thiol-ene reaction was reported as early as 1926, in which it was found that allyl mercaptan could polymerize upon heating44. However, it has been the free radical thiol-ene addition of thiols to electron-rich/electron-poor carbon-carbon double bonds and the catalyzed Michael addition to electron-deficient carbon-carbon double bonds that have received most of the attention during the last century45. In the middle of the last century thiol-ene photopolymerization was used in industrial scale to produce a range of different products, mainly crosslinked systems. However, due to bad odour, yellowing (caused by the residues of the photoinitiators) and the introduction of the cheap acrylic systems, thiol-ene chemistry was to a large extent abandoned. The development of novel catalyst (eliminating the problem of yellowing), the lack of oxygen inhibition

1,4-1,2,3 triazole

R H

R

R2

R'N N

+

N

RC u

C uL L

C uN

NN

C u

R

R'

LL

NN

N

R

R'

R

NN

N

R

R'

R H

NN

N

R

R'

C u2Ln

B B-H

C u2Ln

C u2Ln

C u2Ln

C u2Ln

B -H

B

C u2Ln [C u2Ln]2

Scheme 2.2. Mechanism of the CuAAC reaction producing the 1,4-1,2,3 triazole14.

8

2 INTRODUCTION

and reduced toxicity compared to acrylic systems as well as being a step growth rather than a addition polymerization has lead to a rejuvenation of the thiol-ene chemistry46, 47. Due to its efficient nature, the term click chemistry was recently (2008) also applied to the traditional thiol ene-reaction48. The use of thiol-ene click chemistry has exploded and there are already a number of reviews on the subject12, 45, 49, 50.

The step growth mechanism of the thiol-ene reaction proceeds in a similar fashion irrespectively the reaction is photoinitiated or a base catalyzed Michael reaction (Scheme 2.3), the only difference being the nature of the propagating species (either radical or anionic)45, 46. Although the reaction is not inhibited by oxygen, the presence of oxygen may alter the route and the final product (Scheme 2.4)46, 47. Thus, when performing thiol-ene click reactions where absolute purity of the product is necessary (e.g. dendrimer synthesis) removal of oxygen prior to the reaction is preferred, whereas in the case of film formation this side reaction is often neglected.

R S H R S S C R'R

R'

R S H

SR'R

+ Initia tor

P ropagation

C hain trans fer

Scheme 2.3. Idealized free radical thiol-ene reaction which also applies for Michael addition thiol-ene reactions if the radicals are replaced with anionic counterparts.

R S R'

S C R'R

R S S

R'R

R S H

O 2

SR'R

OO

R S HS

R'R

OO H

R S

+

+

+

Scheme 2.4. Oxygen scavenging mechanism for free-radical polymerization in the presence of aliphatic thiols.

9

2 INTRODUCTION

The structure of the ene is of significant importance for the reaction rate46. For radical initiated thiol-ene systems the reaction rate generally increases with increasing electron density, although this does not apply to norbornenes, methacrylates, styrenes and conjugated dienes where other factors influence to a great extent. In general the following relative reaction rates apply:

norbornene > vinyl ether > propenyl ether > alkene ≈ vinyl ester > N-vinyl amides > allyl ether ≈ allyl triazine ≈ allyl isocyanurate > acrylate > unsaturated ester >N-substituted maleimide > acrylonitrile ≈ methacrylate > styrene > conjugated dienes.

The relative reaction rate of thiols has not been investigated to the same extent but studies show that alkyl 3-mercaptopropionates and alkylthioglycolates have a higher rate of reaction than alkyl thiols46. Michael addition reactions between thiols and enes can take place between thiols and electron–deficient enes in the presence of bases, metals, organometallics, and Lewis acids45, 49, 50. Enes capable of Michael addition thiol-ene reactions include (meth)acrylates, maleimides, α,β-unsaturated ketones, fumarate esters, acrylonitrile, cinnamates, and crotonates.

As for network formation, thiol-ene systems have several advantages over acrylate based systems including the afore mentioned lack of oxygen inhibition but also a delayed gel point, inherent from the step growth mechanism, and lower glass transition temperature (Tg). The delayed gel point results in higher monomer conversions as the mobility is maintained further in the curing process compared to acrylic systems.

2.3.2 Architecturally advanced polymers

Apart from the conventional polymers including different types of linear and branched structures (Figure 2.2), dendritic polymers and linear dendritic hybrids have emerged. These two classes of polymers represent architecturally advanced polymers and are described in the following sections.

2.3.2.1 Dendritic polymersThe family of dendritic polymers is a class of highly branched polymers

and consists of four main subgroups; hyperbranched polymers, dendrimers, dendronized polymers and dendrigrafts (Figure 2.3)51. The concept of preparation of these materials is similar but the control over the structure differs to a great extent. Hyperbranched polymers and dendronized polymers are statistical polymers and typically have polydispersity indices (PDIs) from 1.1 to 10 while dendrigrafts are considered semi-controlled and typically show

10

2 INTRODUCTION

Monomers

Star polymers

Branched polymers

Comb polymers

Random copolymer

Block-copolymers

Linear polymers

Figure 2.2. Conventional polymeric arcitechtures.

DENDRITIC POLYMERS

DendrimersDendrigrafts

Hyperbranched polymers

Dendronized polymers

Figure 2.3. Family of dendritic polymers.

PDIs between 1.1 and 1.551, 52. Dendrimers on the other hand are perfectly branched materials and are monodisperse.

11

2 INTRODUCTION

Architecturally, dendrimers consist of three distinct regions; the core, the interior and the end-groups (Figure 2.4). To the core of the dendrimer, branched wedges, so called dendrons, comprising of generations (layers) of ABX monomers, are attached. The end-groups are situated at the periphery and the part between the core and the periphery is called the interior. Owing to their architecture, the nanosized dendrimers of higher generations are globular and have low intrinsic viscosity, as well as a high number of end-groups available for further functionalization.

Dendron

Peripheral end-groups

Core

B B

A

AB2 monomer

1st

3rd

2nd

4th

Generation

Dendrimer

Figure 2.4. Dendrimer arcitechture displaying the different architectureal features as well as the AB2 monomer.

Two main synthetic approaches for synthesis of dendrimers exist; the divergent approach and the convergent approach51, 53. In the divergent approach the core is the starting point for the synthesis from which generations of the dendrimer are grown outward. In the convergent approach the periphery is the starting point from which dendrons are grown and finally attached to the core. So far the divergent approach has proved most successful and dendrimers synthesized by this approach include poly(amidoamine) (PAMAM)54, 55,

12

2 INTRODUCTION

poly(propylene imine) (POPAM)56, 57 and 2,2-bis(methylol) propionic acid (bis-MPA) dendrimers58-63. An example of a dendrimer synthesized by the convergent approach is the aromatic poly(benzyl ether) dendrimer64, 65. The iterative synthetic procedures typically require an activation/deprotection step before the growth of the next layer, and the synthesis and purification is often tedious. However, recent advances have shown that it is possible to reduce the number of synthetic steps using orthogonal chemistries (e.g. CuAAC and esterification reactions)41, 66-72.

Biomedical applications is one of the main areas of potential application of dendrimers, since high cost usually can be tolerated if significant advantages are obtained. For biomedical applications, the biocompatibility of the material is of outmost importance, especially for in vivo applications, and the commercially available bis-MPA dendrimers has proven to be a leading candidate as they are biologically non-toxic and non-offensive73, 74.

2.3.2.2 Linear dendritic hybrid materialsCombining the traditional linear polymers with dendrimers has given birth to

the concept of linear dendritic hybrid materials75. The hybrids can be combined into a number of different architectures e.g. DL, LDL, LDn, LnD, (LD)n, L(LD)n etc, where L denotes a linear segment and D a dendritic segment. Two illustrative examples are shown in Figure 2.5, i.e. DL and L8DL. Applications including nano-reactors, catalysis, drug delivery, and other biomedical applications have emerged as promising areas of interest for the linear dendritic hybrids. In order to synthesize linear dendritic hybrid materials, and to fully comprise the advantages of monodisperse dendrimers/dendrons, the use of controlled polymerization techniques is required. This enables the tailoring of precise (but not monodisperse) architectures of unlimited variety. Utilizing the ambivalent structure of linear dendritic hybrids is also an excellent approach to tailor novel amphiphilic structures.

DL L8DL

Figure 2.5. The linear dendritic hybrid structures dendritic-linear (DL) and linear8-dendritic-linerar (L8DL).

13

2 INTRODUCTION

2.3.3 Ring-opening polymerization of cyclic esters

As stated earlier, the control of the structure when designing macromolecules is of outmost importance, thus the use of controlled polymerization techniques is required. Two of the most popular classes of controlled polymerization techniques are the controlled radical polymerization (CRP) and the ring-opening polymerization (ROP) techniques. When designing materials for biomedical applications additional requirements are put on the materials, such as biocompatibility and biodegradability. The CRP techniques produce polymers with purely carbon based backbones which impairs biodegradability. In difference, products of ROP of cyclic esters yield polymers that are biocompatible and degradable either through hydrolytic or enzymatic degradation76, 77. The most common monomers include lactide and ε-caprolactone yielding the poly(lactide) PLA (or poly(lactic acid)) and poly(ε-caprolactone) respectively (Figure 2.6).

O

O

OO

O

O

OO

OO

-caprolactone ( -CL) poly( -caprolactone) (P( -CL) or PCL)

lactide polylactide (PLA)

Figure 2.6. Structure of the common cyclic monomers ε-caprolactone and lactide and their respective polymers.

A number of different ROP techniques is reported in the literature, they include anionic, coordination, cationic and activated monomer polymerizations as well as polymerizations in dispersed media78. The most utilized technique for ROP is the coordination polymerization using tin(II) 2-ethylhexanoate or tin octoate (Sn(Oct)2). It is effective, versatile, commercially available, soluble in most organic solvents and lactones, and is approved by the Food and Drug Administration (FDA)76, 78, 79. The mechanism76, 78 (Scheme 2.5) has, after many years of study and debate, been attributed to a coordination mechanism

14

2 INTRODUCTION

starting with the formation of the initiator from Sn(Oct)2 together with a co-initiator (usually a hydroxyl or amine). During the initiation and propagation the catalytically active tin(II) alkoxide opens and inserts the monomer thus forming the polymer. The active alkoxide may subsequently transfer to another chain or the octanoic acid. Possible side reactions include esterification with octanoic acid, transesterification and macrocyclization.

Owing to its mechanism, the complete removal of Sn(Oct)2 is extremely difficult79 and although approved by the FDA, the toxicity of Sn(Oct)2 can be a cause of concern. Hence the development of organic catalysts80 and enzymes81 for ROP are areas of great interest. In addition, the presence of residues of metal catalysts is an issue of great concern also for applications in micro-electronics82.

ROP is also recognized as an efficient tool for the preparation of linear dendritic hybrids, typically dendrons bearing hydroxyl or amine functionalities at the periphery have been used as macroinititors75.

ROP of cyclic esters for the preparation of linear dendritic hybrid materials has proven very efficient. A few of the early and original publications include the synthesis of DL structures, based on benyl ether dendrons and PCL, by Gitsov et al.83 and the synthesis of DLn structures, based bis-MPA dendrons and PCL, by Trollsås et al.84. More recently Hua et al.85 reported on the synthesis of amphiphilic LDLn structures based on PEG, PAMAM dendrons, and PCL.

Sn(O ct)2 O ctH

O ctH

O ctSnO RR O H

O ctSnO R R O SnO RR O H

O

O

S nO

R X

S nO

OR

O

Xn

R O H

O ctH

HO

OR

O

n

HO

OR

O

n

XSnO R

XSnO ct

+ +

+ +

X = O c t or R O

n

+

+

Scheme 2.5. Coordination mechanism of Sn(Oct)2 for ROP of ε-caprolactone76, 78.

15

2 INTRODUCTION

2.4 Applications

The different materials dealt with in this thesis have different areas of intended applications. The hydrogel network structures are intended for marine antifouling whereas the linear dendritic hybrids are mainly aimed for the production of isoporous membranes and micelles for drug delivery. This section is devoted to the background of these areas of application.

2.4.1 Marine antifouling

2.4.1.1 BiofoulingMarine biofouling can be defined as the undesirable accumulation of

microorganisms, plants, and animals on artificial surfaces immersed in sea water86, 87. Ever since man started to travel the waters, marine biofouling has been an issue of concern as it increases the roughness of the hull, thus the friction, leading to reduction of speed and maneuverability. The increased resistance due to fouling can be up to 80%, causing a massive increase in fuel consumption88. As an illustrative example a high-speed containership having 30% extra drag resistance will suffer a speed loss of 1.8 knots and consume 70 tons more fuel per day88. Bearing this in mind it is easy to see why so much effort has been put into the development of antifouling coatings.

In order to develop an antifouling coating with adequate performance it is important to understand the underlying fouling mechanisms and the organisms behind it. The following timeline has been established for the progress of fouling (Figure 2.7)86, 87, 89, 90. Within minutes of immersing a surface into a marine environment organic macromolecules such as proteins, polysaccharides and proteoglycans will adsorb to the surface by e.g. electrostatic or hydrophobic interactions (I). This layer acts as primer for the subsequent colonization by microorganisms such as bacteria and diatoms, which occurs within 1-24 h (II). If allowed to grow, these organisms will within a week form a biofilm further protecting them from predators and toxins. This biofilm will also attract spores of macroalgae and protozoa that further colonize the surface (III). After approximately 2-3 weeks, larvae of macroorganisms, such as different barnacles, mollusks, and tunicates are attracted (IV). Once the macroorganisms have colonized the surface they will contribute to the largest increase in surface roughness thus drag resistance.

16

2 INTRODUCTION

2.4.1.2 Development of antifouling technologiesThe battle against marine biofouling is, as mentioned, by no means a new

battle. The oldest known attempts to prevent marine biofouling dates back to 700 B.C. and the Phoenicians and Carthaginians who used wax, tar, asphalt, pitch and copper sheeting to protect their wooden ships91. Up until the mid 19th century variations of these methods were the main strategies for antifouling. In the mid 19th century the first paints for marine use were developed consisting of a binder and a biocide. Binders, such as linseed oil, Shellac varnish, tar and other resins were used. Toxic pigments used included, copper, arsenic and mercury oxides. These coatings provide a fair protection against fouling organisms and further improvement was achieved with the organo-metallic acrylic paints developed in the 1950s90, 91. During the second half of the 20th century three main types of marine antifouling coatings have been dominating the market; soluble matrix paints, insoluble matrix paints and self-polishing paints. The different types of coatings are well described in recent publications and the reader is directed to these for further reading87, 90, 92. The most efficient antifouling coatings, so far, are the self-polishing coatings containing tributyl tin (TBT-SPC), introduced in 1977. The TBT-SPCs are especially efficient when a co-biocide, such as copper oxide, is added, since green algae and some diatoms are tolerant to TBT. It was, however, not long until the first deleterious effects of TBT were seen and in busy harbors TBT accumulated into toxic levels. As TBT accumulated up the food chain it caused a range of syndromes including dysfunction of calcium homeostas and inhibition of certain functions in mitochondria and chloroplasts87, 90, 92. Consequently, a total ban was enforced by the International Maritime Organization (IMO) starting January 1st 200893.

I II

III IV

macromolecule microorganism

spore of macroalgae larvae of macroorganism

Figure 2.7. The different stages of fouling.

17

2 INTRODUCTION

2.4.1.3 Non-toxic antifouling strategiesSince TBT was banned, and many other biocides, including the common

CuO-pigments, have come under debate, several non-toxic strategies are currently evaluated. Brady94 has listed a few key parameters that a polymer surface should have in order to have an adhesion-resistant surface:

A flexible, linear backbone which introduces no undesirable interactions;

a sufficient number of surface-active groups which are free to move to the surface, there to impart a surface energy in a desired range;

low elastic modulus;

a surface which is smooth at the molecular level to avoid infiltration of a biological adhesive leading to mechanical interlocking;

high molecular mobility in the backbone and surfaceactive sidechains;

a thickness which controls the fracture mechanics of the interface;

molecules which combine all of the above factors, and are physically and chemically stable for prolonged periods in the marine environment.

One type of coatings that fulfill many of these requirements is the silicone based antifouling coatings95. They work by the principle of low surface energy making attachment to coatings difficult and perform well for several years especially for high speed vessels (≥22 knots) and vessels sailing at 15-30 knots having short idle times90. The silicone based coatings are also the, so far, only commercially successful non-toxic antifouling coatings. Other potential technologies yet to find commercial success include fluorinated coatings, smart polymers, fibre coatings, biocide-free self-polishing coatings, scrubbable and inert coatings, and hydrophilic coatings96.

Hydrophilic coatings are of certain interest as it is known from the biomedical field that hydrophilic coatings can resist protein adsorption97, i.e. the first step of the fouling mechanism as stated above. By avoiding the first step it is anticipated that the whole fouling process can be stopped at its very beginning. Research on hydrophilic coatings for marine antifouling have mainly been performed on self-assembled monolayer’s (SAMs)98 and hydrogels96. Hydrogels are especially interesting as they allow tailoring of the elastic modulus indifferent to SAMs. So far there are only a limited number of publications dealing with hydrogels for marine antifouling. For example poly(hydroxyethyl methacrylate) (PHEMA) hydrogels have been evaluated using field tests. However, without the addition of biocides the coatings had poor antifouling99, 100. In other studies, a number of different hydrogels including alginate, chitosan, poly(vinyl alcohol), and

18

2 INTRODUCTION

agarose hydrogels were evaluated in laboratory tests with barnacles and marine bacteria101, 102. It was concluded that all tested hydrogels had lower settlement than polystyrene surfaces, and that the differences between the gels were due to inherent chemical differences in the polymer network rather than variation of modulus and hydrophilicity. A few publications dealing with copolymers comprising PEG have been published. Copolymers comprised of PEG and hyperbranched fluoropolymers displayed adhesion resistance to a range of biomacromolecules as well as inhibiting the settlement of zoospores and the marine algae Ulva103. In another study, a copolymer hydrogel consisting of PEG and PHEMA displayed promising results to a range of different fouling species104.

2.4.2 Isoporous membranes

By controlling the organization of polymers on a surface it is possible to form isoporous structures also known as honeycomb membranes. Depending on the size of the pores, these structures have found present and potential applications such as picoliter beakers, lotus leaf structures, substrates for cell growth, optical band gap materials, inverse opals, porous conjugated materials, materials for sensor applications, energy transfer materials and materials for photovoltaic applications105.

The condensation of moisture in the air to form water droplets on cold surfaces and their ability to form hexagonal arrays has been known for about a century106. The so-called breath figures were, however, not stable over time and it was not until 1994 that the first stabilized breath figures were reported107. François and coworkers discovered that star block-copolymers of polystyrene and poly(p-phenylene) dissolved in CS2 and casted under humid conditions self-organized into hexagonal arrays. Although the mechanism is not fully understood a simplified mechanism, as seen in Figure 2.8, involves evaporative cooling of the volatile solvent, condensation of water droplets, stabilization of the condensed water droplets and pore formation105, 108-110

Evaporation of solventCondensation of water droplets

Isoporous membrane

Figure 2.8. Formation of an isoporous membrane by the breath-figure technique.

19

2 INTRODUCTION

Isoporous membranes using the breath figure technique have yielded pores from 50 nm to 200 μm, where the pore size is highly dependent on the casting conditions105. Factors affecting the pore size include: polymer concentration, air flow, relative humidity, molecular weight and architecture. The pore size (PS) varies with concentration (C) as PS C-1. Higher rate of air flow (AF) will affect the pore size as PS -AF and relative humidity (RH) affects the pore size as PS RH. The effect of molecular weight and architecture is more complex but it has been observed that star shaped polymers will form an isoporous membrane more readily than a linear equivalent105. A thorough review on the subject was written by Bunz105 in 2006 and the reader is referred to this publication for a more thorough elucidation on the effect of different parameters.

Linear dendritic hybrids have successfully been used for the formation of isoporous films111. In this study, a star polymer of polystyrene was peripherally functionalized with dendrons of different generation ((DL)n) and end-groups functionalities were investigated. It was found that both the choice of the end-group and the generation had a profound effect on the pore morphology and size.

2.4.3 Drug delivery

Another interesting application for amphiphilic polymers lies within polymer therapeutics 112 due to the enhanced permeability and retention effect (EPR)113 of tumors. The EPR implies that tumors have hyperpermeable angiogenic vasculature, allowing the penetration of macromolecules and micelles, in combination with poor lymphatic drainage. This causes an up to 70-fold passive increase of concentration of such particles in tumor cells compared to the systematic concentration when the particles are administrated intravenously.

If an amphiphile is present in a high enough concentration it will form a micelle. The lowest required concentration to form a micelle is known as the critical micelle concentration (CMC). In an aqueous system, such as the human body, the hydrophobic part of the amphiphile will be present at the core of the micelle whereas the hydrophilic part will be at the outer parts (Figure 2.9). This implies that a hydrophobic drug may be incorporated into the core of the micelle114. The rate of drug release is then diffusion controlled, allowing a controlled release of the drug. An important advantage of polymeric micelles is that their CMC is rather low compared to micelles based on molecules of low molecular weight. Thus, when administering a micelle based drug delivery system (DDS) comprising of polymeric surfactants, a lower concentration can be administered, without causing disruption of the micelle upon dilution in the

20

2 INTRODUCTION

blood pool. Micelle based DDSs have several advantages as it alters the pharmacokinetics

of a drug to a great extent enabling controlled release and extended circulation times of the drug (Figure 2.10)115, 116. This implies that a higher dose can be given while minimizing the side effects of the drug, at the same time increasing the drug availability. In addition, the micelle provides a stealthy cloak for the drug hindering its clearance by the liver. The prolonged circulation time also provides increased effect of targeted drugs as more of these will be able to pass the targeted site. All these effects in combination with the afore mentioned EPR affords a promising future for micelle based polymer therapeutics.

An ideal micelle for pharmaceutical applications should have a CMC in the low millimolar region, have a size between 10-100 nm and have a loading efficiency of about 5-25 wt%114. In addition, it should be stable enough to stay in the body for an extended time and eventually degrade into non-toxic products that can be cleared from the body. The hydrophobic/hydrophilic balance is of outmost importance and generally CMC is decreased with increased relative weight of the hydrophobic part117. However, this only applies to materials having the same hydrophobic and hydrophilic components since the chemistry of the blocks have a large impact on the CMC. Another important factor affecting the final properties of the micelles is the architecture. This parameter has, however, not yet been fully elucidated and further studies, solely varying the architecture, are required118, 119. The most common hydrophilic block is PEG due to its non-toxic nature and stealth function. The hydrophobic block has, on the other hand, been varied to a larger extent, although biodegradable polymers such as aliphatic polyesters are preferred112, 114, 117.

Below CMC Above CMC

hydrophilic

hydrophobic

Figure 2.9. Amphipilic polymers in solution below and above the CMC.

21

2 INTRODUCTION

Toxic level

Minimum effect level

Time

Dru

g co

ncen

tratio

n

Unsafe dose

Safe dose

Controlled release

Figure 2.10 Drug concentration as a function of time using different doses and types of administration. The safe dose (∙∙∙), and the unsafe dose (- - -) with direct administration of the drug, compared to a DDS displaying controlled release (—)115.

23

3 EXPERIMENTAL

3. EXPERIMENTAL

3.1. Materials

Tetraethylene glycol dimethacrylate (technical, >90%) (1T) and poly(ethylene glycol) dimethacrylate (Mn ≈ 750) (1P), N,N’-dicyclohexylcarbodiimide (DCC), monomethoxy poly(ethylene glycol)s with molecular weight 2000 and 5000 g∙mol-1, 1,3,5-triallyl-1,3,5-triazinane-2,4,6-trione (98%), albumin bovine - fluorescein isothiocyanate conjugate (BSA-FITC), pyrene (puriss), CaH2 (95%), NaN3, N,N-diisopropylethylamine (99.5%) (DIPEA), and propargylalcohol (≥99%) were purchased from Sigma-Aldrich. CH2Cl2 (p.a), tetrahydrofuran (THF) (p.a.), methanol (p.a.), heptane (p.a.)), and ethyl acetate (EtOAc) (EMPROVE exp), dimethyl sulfoxide (DMSO) (for synthesis), Et3N (for synthesis), and toluene (Seccosolv) were acquired from Merck. Ethanol (EtOH) (96%), acetone (anhydrous), pyridine (AnalR NormaPur), and THF (unstabilized, HPLC) were purchased from VWR. Diethyl ether (analytical), and MeOH (analytical) were purchased from Fisher Scientific. Allyl bromide (99%), acetone (spectrophotometric grade), and DOWEX were acquired from Alfa Aeaser. MgSO4 and silica gel for flash chromatography, KOH and benzene (pro analysi) were purchased from Acros. Tris(2-(3-mercaptopropionyloxy)ethyl) isocyanurate (4) was purchased from Wako and thiocure ETTMP 700 (6) was supplied by Bruno Bock. Bis-MPA was supplied by Perstorp and the alkyne functional dendrons (alk-Gn) were purchased from Polymer Factory Sweden AB. The photoinitiators 1-hydroxy-cyclohexyl-phenyl-ketone (Irgacure 184) and 2,2-dimethoxy-1,2-di(phenyl)ethanone (DMPA) were supplied by CIBA. ε-Caprolactone (Acros) was distilled over CaH2 and stored over 4 Å molecular sieves under Ar (g). Tin(II) 2-ethylhexanoate (95%) (SnOct2) (Sigma Aldrich) was dried using 4 Å molecular sieves in a solution of toluene prior to use. Artificial seawater (ASW) was prepared from Instant Ocean (Aquarium Systems) and Lefant Epoxy Primer (base and hardener) was supplied by Lotréc AB. 4-(Dimethylamino) pyridinium 4-toluenesulfonate (DPTS) was prepared as reported elsewhere120. Acetonide protected bis-MPA anhydride was synthesized as described previously61. Cu(PPh3)3Br was synthesized as described previously121. PEG2k-N

3 and PEG5k-N

3 were synthesized as reported earlier122.

24

3 EXPERIMENTAL

3.2 Instrumentation

Nuclear Magnetic Resonance (NMR) 1H and 13C was performed on a Bruker Avance 400 MHz instrument using the solvent signal or TMS as internal reference.

Infrared spectroscopy (IR) spectra were recorded on a Perkin-Elmer Spectrum 2000 FTIR equipped with a heat-controlled single reflection attenuated total reflection (ATR) accessory from Specac Ltd.

Raman spectroscopy were acquired for all samples using a Perkin–Elmer Spectrum 2000 NIR FT-Raman instrument. Each spectrum was based on 32 scans using 1500 mW laser power.

Size exclusion chromatography (SEC) using THF as mobile phase (1 ml∙min-

1) at 35°C was performed on a Viscotek TDA model 301 equipped with 2 T5000 columns, a VE5200 GPC autosample, a VE1121 GPC solvent pump and a VE5710 GPC degasser (all from Viscotek/Malvern). Conventional calibration using linear polystyrene standards and triple detection calibration using a single linear polystyrene standard were used and the samples were evaluated using OmniSEC 4.5 software.

Differencial scanning calorimetry (DSC) was performed on a Mettler Toledo DSC820 using a heating/cooling rate of 10 °C∙min-1 under N2 atmosphere.

Matrix assisted laser desorption ionization – time of flight mass spectroscopy (MALDI-TOF MS) was performed on a Bruker Uniflex MALDI-TOF MS with SCOUT-MTP Ion Source (Bruker Daltonics) equipped with a N2-laser (337 nm), a gridless ion source and reflector design. 9-Nitroanthracene or dihydroxybenzoic acid was used as matrix with added sodium trifluoroacetate. The obtained spectra were analyzed using FlexAnalysis (Bruker Daltonics).

Static contact angle measurements were performed on a KSV Instruments CAM 200 equipped with a Basler A602f camera, using 5 μl droplets of MiliQ water and a relative humidity of 50%. Determination of the contact angles were performed using CAM software.

Fluorescence spectroscopy for protein adsorption measurements was performed using a Zeiss Axiplan 2 imaging equipped with a CCD-camera and AxioVision 4.7 software.

Dynamic light scattering (DLS) experiments were performed on a Malvern ZEN 3600 Zetasizer Nano ZS operating at 633 nm and 25°C.

Transmission electron microscopy (TEM) analysis was performed on a JEM-2010/INCA OXROD TEM (JEOL/OXFORD) at a 200 kV accelerating voltage. Samples were prepared by applying micelle solutions to plasma etched carbon coated copper grid. Using filter paper excess solution was wicked away and the samples were stained using uranyl acetate (saturated solution in H2O).

25

3 EXPERIMENTAL

Optical microscope images were recorded on a Leica DMIRM optical microscope.

Field emission – scanning electron microscopy (FE-SEM) images were aquired using a Hitatchi S-4300 FE-SEM on previously gold sputtered surfaces.

Fluorescence spectrocopy for CMC measurements were performed on a Varian Cary Eclipse collecting emission spectra using an excitation wavelength of 332 nm.

UV-Vis spectroscopy was performed on a Varian Cary 300 Bio using a wavelength of 380 nm.

3.3 Synthetic, preparative and analytical procedures

This section describes some of the general synthetic, preparative and analytical procedures. For a more detailed description, the reader is referred to the respective articles.

3.3.1 Hydrogel coatings

3.3.1.1 Preparation of thiol functional glass surfacesMicroscope slides with cut edges (76 x 26 mm, Thermo Scientific, Menzel-

Gläser) were cleaned by immersion in household detergent, Yes (Procter & Gamble). To further clean the slides they were immersed in a solution of HCl:H2O2:H2O (1:1:5). The slides were rinsed with deionized water and EtOH followed by immersion in EtOH, where they were stored until further use. In order to avoid detachment of the coatings, the slides were functionalized with thiol groups. The slides were submerged for 10 min in a 1:1 H2O:EtOH solution containing 0.05% glacial acetic acid and 0.4% 3-mercaptopropyl trimethoxysilane followed by air drying in a LAF bench. To cure the silane, the slides were placed in an oven at 115°C for 10 min followed by immersion in EtOH. Before coating, the slides were wiped using Durx® 670 cleanroom wipers soaked in EtOH followed by thorough rinsing with EtOH and air drying in a LAF bench.

26

3 EXPERIMENTAL

3.3.1.2 General coating procedure for hydrogel coatings H1-H8 on glass slides Equivmolar amounts of methacrylates/allyls and thiols with a total dry

content of 3.2 g were weighed up and diluted with 400 mg of butyl acetate. 0.5 wt% of Irgacure 184 was added and the solution was mixed to a homogeneous solution using a vortexer. The dry slides were subsequently coated using an Ericsen applicator with a gap of 60 μm, with each 3.2 g batch yielding 10-15 coated slides. The coated slides were left to flash off the solvent for 1 h before UV-induced polymerizations were performed using a Fusion UV Curing System Model F300 equipped with Fusion electrodeless bulbs standard type BF9 (Lamp power 300 W per inch, 1800 watts total). The films were cured by 15 successive passes under the lamp to give a total dose of 500 mJ∙cm-2 as determined by measuring the intensity with a UVICURE® Plus from EIT Inc., Sterling, VA, USA.

3.3.1.3 Application of primers on PVC panelsNon-plasticized PVC panels with a size of 10x10 cm were prepared with a

commercial primer. Prior to application of the primers, the panels were sanded using a 180 paper. A 2K epoxy primer (EP) was applied twice using a brush according to the instructions from the manufacturer.

3.3.1.4 Application of hydrogel coatings on primed PVC panelsThe unsaturated PEGs and the thiol crosslinkers were mixed in equimolar

amounts, in butyl acetate (BuOAc) to give a dry content of 90% and 0.5wt% of photoinitiator (Irgacure 184) was added. The solution was thoroughly mixed using a vortexer followed by application of the coatings to primed PVC panles using a sponge. The coated panels were left to flash off the solvent for 1 h before UV-induced polymerizations were performed using a Fusion UV Curing System model F300 equipped with Fusion electrodeless bulbs standard type BF9 (Lamp power 300 W per inch, 1800 W total). The films were cured by 15 successive passes under the lamp to give a total dose of 500 mJ∙cm-2 as determined by measuring the intensity with a UVICURE Plus from EIT Inc., Sterling, VA.

3.3.1.5 Swelling and stability studies Gravimetric and contact angle measurements were performed to evaluate

swelling and stability of the coatings. The gravimetric studies were performed by weighing the glass slides before and after coating formation to determine the dry weight of the films. Three slides of each coating system were subsequently submerged in deionized water (DIW) and ASW respectively. After 1 day of submersion, the slides were taken up, gently dried and weighed after which

27

3 EXPERIMENTAL

contact angle measurements were performed. This procedure was repeated on day 2, 3, 4, 9, 15 and 28. (On day 15 only gravimetric analysis were performed as the contact angle equipment was out of order).

3.3.1.6 Protein adsorption studies Protein adsorption studies were performed after swelling the coatings in 25

ml of phosphate buffer solution (PBS) for 1h. The coatings were subsequently incubated by placing 500 μl drops of 0.1 mg∙ml-1 FITC labeled BSA in PBS on the top of the coatings. After 10 min of incubation, the coatings were rinsed with 20 ml of PBS and immediately imaged using a fluorescence microscope. Duplicate images of each coating before and after incubation with BSA were recorded. The total fluorescence was determined using the built-in FITC filter of the microscope, with a constant exposure time, magnification, and image area for all the surfaces. To calculate the total fluorescence from BSA, the background fluorescence for each coating type was first subtracted using ImageJ software and the total fluorescence was calculated using the built-in plug-in.

3.3.1.7 Bioassays Bacterial assay. The bacterial suspension of the marine bacteria Cobetia marina,

used for the testing was obtained after the cells were repeatedly washed with PBS and centrifuged to remove excess PBS for optimal adhesion.

The conditioned replicate slides were immersed for 1h in polystyrene quadriPERM plates (GreinerBio-one Ltd) containing 8 ml suspension of C. marina bacteria with an OD of 0.2 (595 nm). The slides were incubated on a shaker (150 rpm) for 1 h at 28°C. Non-adhered and loosely attached cells were removed by dipping the slides once in sterile seawater. The slides were transferred back into quadriPERM plates containing 8 ml of sterile SW with added growth medium and incubated again for 4 h at 28°C under gentle shaking (150 rpm). At the end of incubation, the slides were rinsed again and then placed into slide holders and partially air-dried. Attached cells were stained, using the fluorochrome SYTO13 (1.5 mM), for biomass quantification in a Tecan plate reader (GENios, Magellan software) Biomass quantification on acid-washed glass (hydrophilic) and Silastic T2 (hydrophic) was also determined and used as reference substrates to which settlement responses on the coatings could be compared.

To quantify the adhesion strength of attached bacteria, a rotating drum test was used. Slides were treated as above. After the growth step, replicate slides of each coating were rotated on the rotating drum for 10 min at 12 knots in natural seawater. This rotational speed of the drum exposes the bacteria to shear stress (turbulent flow), causing an amount of bacteria to be removed

28

3 EXPERIMENTAL

from the surfaces. The remaining bacteria were then quantified using SYTO13 stain as described above. Data are expressed as % biofilm removal (Δ biomass/ biomass before release x 100%).

One sample of the TNO references and one sample of each coating were exposed without bacteria under the same conditions as the other samples serving as coating blanks to check for autofluorescence and contamination.

Diatom assay. The common fouling species Amphora coffeaeformis was used for this test. Diatom cultures were maintained in the growth room at TNO (18°C and 24 h light exposure) in enriched filtered sterilized seawater with silicate enriched F2 growth medium. 80 μl of 3 - 4 days grown diatom cell suspension was placed in a line over the samples

The samples were incubated for two hours in the dark to allow the cells to attach to the surface. The diatom slides were then gently dip-rinsed with sterile seawater to remove unattached diatoms. Diatom fluorescence on the slides was measured using the Tecan plate reader (GENios, Magellan software). The slides were transferred into quadriPERM plates containing 10 ml sterile, filtered seawater with added growth medium and incubated for 5 days at 18°C with 24 h light exposure. After the incubation period, the slides were removed, gently dip-rinsed in sterile seawater and then the fluorescence was re-measured using the Tecan plate reader. Replicated samples were evaluated and compared with acid washed glass controls and the hydrophobic TNO standard Silastic T2.

3.3.1.8 Field studiesField studies were conducted in the North Sea harbour of Norderney,

Germany. The panels were first exposed on the 27th of May and the field tests proceeded until the 16th of September. During this exposure the panels were inspected once a month and photographed both before and after a dynamic phase. The dynamic phase consisted of the towing the panels attached to a surf board for 30 min at a speed of 5 knots. Fouling rate was determined using ASTM 6990-03 standard where 100 is a perfectly clean surface and 0 is a completely fouled surface.

3.3.1.9 Mechanical properties of coatings Pendulum hardness tests were performed on Erichsen 299/300 pendulum

hardness tester according to ASTM D 4366-95. All tests were performed three times on each coating.

Pencil hardness tests were performed according to ASTM 3363-00. Hardness of hardest pencil used without rupture, visually determined, of the film is reported.

29

3 EXPERIMENTAL

Microindentaion was performed on a CSM Instruments Nano-scratch tester equipped with an indenter with a Berkovich shaped diamond and evaluated using Indentaion software 3. The loading, as well as the unloading rate, was 500 mN∙min-1 with a pause of 15 seconds when the maximum depth of 5 μm is reached. All measurements were performed five times on each coating.

Adhesion was determined according to ASTM D 3359. A lattice was cut with a crosshatch cutter and the lattice was brushed five times back and forth in each diagonal direction. The lattice was subsequently inspected visually and graded from 0-5. Grade 0 being no failure and 5 complete failure.

3.3.2 Linear dendritc hybrids

3.3.2.1 Polymerization of ε-CL using dendrons with alkyne or allyl functional cores as initiators

Dendrons of generation 0 to 4 with alkyne or allyl functional cores were used as macroinitiators for ROP of ε-CL using Sn(Oct)2 as catalyst in toluene at 110°C. The targeted degrees of polymerization (DPs) were set to be reached at 75% monomer conversions. A typical example follows: A flame dried 100 ml round bottom flask equipped with a stir bar was charged with 189 mg (1.10 mmol) of alk-G1-OH. The flask was sealed with a septum and 2 ml of dry toluene was added. After heating to 110°C, the toluene was removed under vacuum for a period of 20 min and refilled with argon, 20 ml of dry toluene and 19.0 ml of ε-CL (175 mmol). Finally, 0.22 ml of a 1.00 mmol∙ml-1 Sn(Oct)2 stock solution (0.22 mmol) were added. The reaction was allowed to proceed until a conversion of 75% had been reached (usually 2-3h), as observed by 1H-NMR. The reaction vessel was removed from the oil bath, diluted with 40 ml of CH2Cl2, and precipitated in 1.5 L of MeOH. The white precipitate was subsequently filtered off and dried under vacuum.

3.3.2.2 Synthesis of amphiphilic linear dendritic hybrid block-copolymers using CuAAC chemistry

Generally, CuAAC reactions for the formation of amphiphilic linear dendritic hybrids had a alk-Gn-PCL:PEG-N3:Cu(PPh3)3Br:DIPEA feed ratio of 1:1.2:0.5:0.25. A typical example follows: A 10 ml round bottom flask was charged 1.00 g (0.081 mmol) of alk-G1-PCL60 and 0.20 g (0.099 mmol) of PEG2k-N

3 and dissolved in 5 ml of THF. This was followed by the addition

of 26 mg of DIPEA and 38 mg of Cu(PPh3)3Br. The reaction mixture was subsequently heated to 40°C and the reaction was allowed to proceed over

30

3 EXPERIMENTAL

night. Using 1H-NMR the conversion of the alkyne group was monitored. When complete conversion of the alkyne group had been reached the product was isolated by precipitation in 100 ml cold MeOH followed by filtration and drying under vacuum.

3.3.2.3 Synthesis of amphiphilic linear dendritic hybrid block-copolymers using thiol-ene chemistry

Generally, thiol ene click reactions for the formation of amphiphilic linear dendritic hybrids had a all-Gn-PCL:PEG-SH feed ratio of 1:3. A typical example follows: A 5 ml round bottom flask equipped with a stir bar was charged with 500 mg (34 μmol) of all-G1-PCL60 and 210 mg (100 μmol) of PEG2k-SH. After the complete dissolution of the materials in THF (unstabilised), a catalytic amount of the photoinitiator DMPA was added and the solution was purged with Ar(g) for 2 min followed by exposure to UV for 60 min using a high pressure mercury lamp (Oriel).

3.3.2.4 Formation of self-assembled nanoparticels from amphiphilic linear dendritic blockcopolymers

The formation of nanoparticles was achieved by dissolving 10 mg of PEG-Gn-PCL in 4 ml of anhydrous acetone. The polymer solution was subsequently slowly added dropwise using a syringe to a 20 ml of PBS buffer under vigorous stirring. The solution was vigorously stirred for 24 h followed by dialysis in 1 L of PBS using a dialysis membrane with 1kDa MWCO. The dialysis was allowed to proceed for 72 h and with three changes of buffer. The four most hydrophilic materials i.e. the (PEG2k-G0-PCL60, PEG2k-G1-PCL30, PEG5k-G1-PCL60 and PEG5k-G2-PCL30) were further analyzed by DLS and TEM. Materials with PCL:PEG molar mass ratio higher than 3.4 were unable to form micelles as they rapidly precipitated in the buffer solution. Prior to analysis, the micelle solutions were filtered using a 0.45 μm nylon syringe filter in order to remove dust and aggregates.

In order to determine the critical micelle concentration the fluorescent probe technique with pyrene was used.

The drug loading/release capacities were evaluated by encapsulation of doxorubicin (DOX). The micelles were loaded using the following procedure: 69 μl of a stock solution, consisting of DOX in CHCl3 (5 mg∙ml-1) and 3 molar equivalents of Et3N vs DOX, was added drop-wise under stirring to a 10 ml vial containing 3 ml of micelle solution (0.38 mg∙ml-1). The vials were subsequently left open over night in order to allow the CHCl3 to evaporate. Free DOX was removed by spin filtration using Amicon Ultra 4 centrifugal filters with a MWCO of 3kDa. UV-Vis spectroscopy was performed on samples diluted 10

31

3 EXPERIMENTAL

times with DMF:H2O 4:1 (ε = 13050 M-1∙cm-1) to determine loading efficiency. Where the loading efficiency is calculated by the quote CDOX,micelle solution/Cmicelles.

3.3.2.5 Formation of isoporous films from amphiphilic linear dendritic blockcopolymers The films were obtained by casting the polymer films under humid

atmosphere. 50 μL solution of PEG-Gn-PCL in benzene at a concentration of 1 g∙l-1 of polymer was casted on a glass substrate (d = 30 mm). The solution was allowed to evaporate at room temperature (20˚C) in a closed humid chamber (90 % relative humidity) leading to the formation of a solid white film. The films were analyzed by optical microscopy and SEM.

33

4 RESULTS AND DISCUSSION

4. RESULTS AND DISCUSSION

This chapter is divided into two sections, 4.1 describes the fabrication of hydrogel coatings and their potential as marine antifouling coatings. 4.2 describes the synthesis of amphiphilic linear dendritic hybrid materials and their potential application as drug delivery vehicles as well as their self-assembly into isoporous membranes.

4.1 Design of hydrogel coatings and their potential as antifouling coatings

A good antifouling coating is of outmost economical and environmental importance. However, most current technologies include the use of biocides, which could lead to effects on non-target organisms. Hence, the development of non-toxic strategies is of great interest. One non-toxic strategy that has received increased attention is the development and use of hydrophilic coatings. Among this class of coatings, hydrogels are very interesting alternatives. Consequently, using efficient and benign thiol-ene chemistry, a library of hydrogel coatings was designed. The library was designed in order to elucidate some of the properties affecting the antifouling performance and the applicability of the coatings.

4.1.1 Synthesis of monomers and crosslinker

The thermal properties, curing chemistry and hydrolytic stability are of great importance for a coating targeted for marine applications. Accordingly, allyl functional PEGs were synthesized and compared with the commercially available methacrylated PEGs 1T and 1P (T denoting n=4 and P denoting n≈14). In order to study the influence of hydrolytically labile esters on the coating properties, two different sets of allyl functional PEGs were targeted; 2T and 2P (bearing esters) (Scheme 4.1), and 3T and 3P bearing exclusively ethers (Scheme 4.2). The ester bearing allylated PEGs, 2, were afforded by reacting either tetraethylene glycol (TEG) or PEG (n≈14) with 4-(2-(allyloxy)ethoxy)-4-oxobutanoic acid (AEOBA) (synthesis of AEOBA is reported in detail in article I) using DCC dehydration chemistry (Scheme 4.1). The purely ether

34

4 RESULTS AND DISCUSSION

Scheme 4.2 Synthesis of allyl ether functional PEGs, purely ether based (3), where n=4 for 3T and n≈14 for 3P.

based materials 3 were synthesized from TEG and PEG (n≈14) in a two step reaction; intitally the hydroxyl end-groups were alcoxylated using NaH, followed by the addition of allyl bromide (Scheme 4.2). Both sets of materials, 2 and 3, were characterized using 1H-NMR, 13C-NMR as well as FT-IR and FT-Raman. The spectroscopical techniques confirmed the presence of allyls (C=C stretch at 1638 cm-1) and carbonyls (C=O stretch at 1717 cm-1) in 2 and the presence of allyls (C=C stretch at 1645 cm-1) in 3.

OO n

B r

O HO n

HNa HTHF 0 °C

3

Scheme 4.1 Synthesis of allyl ether functional PEGs bearing esters (2), where n=4 for 2T and n≈14 for 2P.

OO

O

O

OO

O

n O

O

O

OO

OO

O H

O HO n

H

D M AP , D P TS , D C C , C H 2C l2, 0 °C

2

To produce an exclusively ether-based network, an ester-free analogue of the tris(2-(3-mercaptopropionyloxy)ethyl) isocyanurate (4) cross-linker was synthesized using a two step, one-pot synthesis. As the first step, 1,3,5-triallyl-1,3,5-triazinane-2,4,6-trione and thioacetic acid were reacted using thiol-ene chemistry followed by hydrolytical cleavage of the thioester to afford the thiol-functional product, 1,3,5-tris(3-mercaptopropyl)-1,3,5-triazineane-2,4,6-trione (5) (Scheme 4.3). The product was isolated using flash column chromatography and analyzed by 1H-NMR, 13C-NMR. FT-IR and FT-Raman analysis of the product further confirmed the study showing absorptions at 1755 cm-1 corresponding the C=O stretch of the carbonyl in the triazine ring and 2568 cm-1 corresponding to the S-H stretch.

35

4 RESULTS AND DISCUSSION

N

N

N

O

OO

S

S

SO

O

O

N

N

N

O

OO

SH

O

N

N

N

O

OO

S H

SH

S H

AIB N, 6 0 °C

HC l

M e O H, 6 0 °C

5

Scheme 4.3 Synthesis of ester free triazine cross-linker (5).

4.1.2 Hydrogel coating preparation, curing and physical properties

Eight different hydrogel systems, H1-H8, were formulated to have a dry content of 90% in butyl acetate (BuOAc) (Scheme 4.4 and 4.5). The different systems were formulated to allow an elucidative study of different structural parameters. Short PEG chains (n=4) were incorporated into systems H1, H4, H5 and H7 whereas longer PEG chains (n≈14) were incorporated into systems H2, H3, H6, and H8. The effect of curing chemistry could be studied by comparing the methacrylate based systems H1-H4 to the allyl systems H5-H8. Systems H1-H6 all contain hydrolytically labile esters whereas H7 and H8 are purely ether based. In order to study the effect of the cross-linker, the rigid crosslinker 4 were incorporated into systems H1-H2 and H5-H6, whereas H3-H4 were formulated with the more flexible 6. The purely ether based systems H7-H8 contain a ester free analogue (5) of 4.

As models substrates, microscope slides were chosen for the fabrication of the hydrogels coatings. In order to promote the adhesion of the coatings, the slides were thiolated using 3-mercaptopropyl trimethoxy silane. All systems wetted the slides well except H5 and H6; however, given a few seconds smooth films were achieved. After allowing the BuOAc to flash off, the coatings were cured by UV with a total dose of 500 mJ∙cm-2. H2 and H4-H6 produced smooth, even, and optically clear coatings apart from H1 and H3 which had slight top curing, and systems H7 and H8 displayed shrinkage. The freshly prepared films were subsequently analyzed using FT-IR and FT-Raman (Figure 4.1). The combination of these techniques is very useful as FT-Raman gives strong signals for thiols and enes, allowing the degree of curing to be determined. FT-IR on the other hand, gives strong signals for carbonyls, hydroxyls and carboxylic acids, making it useful for studies of hydrolytical stability and degradation.

36

4 RESULTS AND DISCUSSION

O

OO

O

n

N

N

N

O

OO

O

O

O

O

O

O

SH

SH

SH O

O

O

S

N

NN

O

O

O

O

O

O

O

O

O

S

S

O

O

O

O

n

OO

O

O

n

n

1T1P

4

H1H2

S O OO

O

n

S

O

O

O

O

S

O

O

O

O

n

O

O

m

O

O

O

m

O

O Om

O

O

OO

O

n

O

OSH

m

O OO

SH

m

O

O

O

SH

m

O

1T1P

H3H4

6

Scheme 4.4. Formation of hydrogel coatings H1 and H2 (A), and H3 and H4 (B).

A

B

37

4 RESULTS AND DISCUSSION

2T2P

4

H5H6

O OO

O

OO

O

n OO

O

S

O

O

O

O

O

O

O

O

O

O

n

S

O

O

O

O

O

OO

OO

O

n

N

NN

O

O

O

O

O

O

O

O

O

S O OO O

OO

O

O

O

n

N

N

N

O

OO

O

O

O

O

O

O

SH

SH

SH

OO n

OSO

n

O

S

On

O

S

O

n

N

NN

O

O

O

N

N

N

O

OO

SH

SH

SH

5

3T3P

H7H8

Scheme 4.5. Formation of hydrogel coatings H5 and H6 (A), and H7 and H8 (B).

A

B

38

4 RESULTS AND DISCUSSION

Raman spectroscopy revealed that all cured thiol-metharcrylate systems had residual thiols remaining, as seen by the peaks at 2573 cm-1 (S-H stretch) (Figure 4.1 and Table 4.1). Some systems had as low thiol conversion as 45% (H1) as seen in Table x. However, the peak at 1683 cm-1, corresponding to the C=C stretch from the methacrylate double bonds, had disappeared completely in systems H1, H2 and H4 with some remaining in system H3 (86% conversion). The Raman spectroscopy thus suggested substantial homopolymerization of the methacrylate groups, which is in good accordance with previous studies46,

123, 124. In contrast, the allylic systems H5-H8 displayed complete conversion of both thiols and allyls as seen in Figure 4.1 and Table 4.1. Only H7 had a small amount of residual thiol suggesting some homopolymerization of the allyls. The relatively high extent of homopolymerization in the thiol-methacrylate systems (H1-H4) is most likely a result of the tight structure rather than differences in reactivity between methacrylate homopolymerization and the thiol methacrylate reaction, as the extent of homopolymerization is reduced with respect to increased length of PEG. This is further supported by the curing behavior of H7 where residual thiols are observed, plausibly as a result of homopolymerization of allyl ethers. In the case of H3, where both thiols and methacrylates remained after curing, the results can be explained with the relatively low concentration of reactive groups as the PEG and the cross-linker are extended and therefore decreasing the reaction efficiency.

For the field study and the mechanical tests, the coatings were applied to 10x10 cm PVC panels primed with a commercial primer (EP). The coating were applied using a sponge and cured using the same procedure as above, resulting in a set of hydrogel coated PVC panels having a coating thickness of about 40-60 μm.

4.1.3 Thermal properties of starting materials and hydrogels

DSC is an efficient tool for the understanding of the intrinsic properties of networks and structural effects of the building blocks on the phase transitions. Hence, DSC was performed on the starting materials as well as the cured films (Table 4.1). PEG structures are normally semicrystalline and their incorporation into cross-linked networks normally inhibits crystallization. Studying the ene terminal PEGs, the effect of the incorporation of ester bonds could clearly be seen. The purely ether based system with the longer PEG-chain 3P displayed the highest degree of crystallinity with a crystallization enthalpy (ΔHc) of 125 J∙g-1. This is considerably high as pure PEG with Mn = 1000 g∙mol-1 has a ΔHc of 165 J∙g-1 125. In addition, the DSC-results show that 3T is the only of the

39

4 RESULTS AND DISCUSSION

600110016002100260031003600

252025402560258026002620 16001650170017501800

H1

H2

H3

H4

H5

H6

H7

H8

(cm-1)

Figure 4.1 FT-Raman spectra of all hydrogel coatings H1-H8 with inset zooms of the thiol absorption region (2620-2520 cm-1) and the carbonyl, allyl and methacrylate absorption region (1800-1600 cm-1).

short PEGs displaying crystallization. This implies that the crystallization is not impaired to any larger extent by the allyl ether end-groups. The introduction of esters has, however, a dramatic effect of the thermal behavior. The crystallization temperature (Tc), the crystallization enthalpy (ΔHc), as well as the melt temperature (Tm) and melting enthalpy (ΔHm) are decreased by the introduction of esters. In fact, in the case of the ester based diallylic PEG 2P, crystallization does not occur during the cooling cycle; instead it shows a glass transition and crystallizes only during the heating cycle, indicating that the crystallization is severely hindered by the ester groups.

From the DSC results of the cured films it is evident that the cross-linking impairs the crystallization of the PEG chains (Table 4.1). Only coating H8, containing the highly crystalline and purely ether based PEG 3P, displays a Tc and a Tm. Furthermore, the effect of hompolymerization is clearly seen comparing the Tgs of the thiol-methacrylate system H1 (Tg = -2.9°C) to its thiol-allyl ether

40

4 RESULTS AND DISCUSSION

Ent

ryPE

Gth

iol

Conv

. SH

(%)

Conv

. C=

C (%

)T g (

°C)

T c (°C

)ΔH

c (J∙

g-1)

T m (°

C)ΔH

m

(J∙g-1

)

Penc

il ha

rdne

ssCr

ossh

atch

adh

esio

nBi

ofilm

relea

se

as %

rem

oval

Dry

Swell

edD

rySw

elled

1T 1P-1

2.3

83.4

9.3

85.6

2T-6

3.9

2P-6

0.7

-42.

0a61

.96.

369

.9

3T-3

8.7

119.

8-2

6.8

125.

5

3P0.

212

4.9

22.0

130.

7

4-4

1.5

5-5

0.9

6-6

5.0

H1

1T4

45>

95-2

.9H

H1

050

H2

1P4

56>

95-3

3.9

HB

2H1

059

H3

1P6

6186

-44.

03H

2H0

129

H4

1T6

73>

95-3

6.4

H4H

11

41

H5

2T4

>95

>95

-24.

12H

3H1

068

H6

2P4

>95

>95

-36.

34H

2H1

119

H7

3T5

88>

95-3

9.4

5H3H

20

H8

3P5

>95

92-5

4.1

25.2

a21

.75.

720

.8>

6H2H

01

H+

glas

s76

silas

tic T

271

EP

2H2H

11

a Cry

stall

izat

ion

occu

rred

dur

ing

the

heat

ing

cycle

.

Tabl

e 4.

1. P

rope

rties

of

hydr

ogel

prec

urso

rs, h

ydro

gel c

oatin

gs, a

nd re

fere

nce

mat

erial

s.

41

4 RESULTS AND DISCUSSION

analogues H5 (Tg = -24°C) and H7 (Tg = -39°C). In fact, all the fabricated thiol-methacrylate coatings display higher Tgs than their allylic counterparts. These results agree well with other studies, where it has been shown that the introduction of thiols in pure methacrylate systems has a lowering effect of the Tg

123, 124.

4.1.4 Swelling and degradation

Since the targeted application for these coatings are within area of marine coatings their swelling and degradation behavior are very important parameters. Hence, the swelling and degradation behavior were evaluated in both DIW (Figure 4.2) as well as ASW (Figure 4.3). To study the coatings, gravimetric measurements were performed on coated glass substrates.

The gravimetric analysis showed that all systems comprising the shorter PEG chains swelled to a less extent than the systems comprising the longer PEG chains. This is expected since shorter chains will form tighter networks with higher cross-linking density. The effect of the swelling medium had a large impact on the behavior of the coatings, and degradation could be clearly observed in ASW, in difference to DIW (Figure 4.4). The effect of having hydrolytically labile esters were also seen, as coatings having esters were more subjected to degradation than the purely ether based coatings, agreeing well with other studies126. The most swollen coating systems (H2 and H8), swelling significantly above 100% in both DIW and ASW, were difficult to analyze for an extended period as they detached from the primed glass slides, as a result of substantial interfacial stresses and hydrolytic degradation of the silane primer.

4.1.5 Mechanical properties

The mechanical properties of an antifouling coating are of outmost importance. However, to define the optimal mechanical properties is not trivial. On one hand, a soft coating is expected to yield improved antifouling94, but on the other hand, soft coatings are more susceptible to abrasion and wear. In order to elucidate on impact of hardness of our coatings, indentation hardness (Figure 4.5), pendulum hardness (Figure 4.6), and pencil hardness (Table 4.1) were used to evaluate the mechanical properties of the hydrogel coatings. As expected, swelled coatings were significantly softer than the dry coatings and longer PEG chains, generally provided a softer coating than a shorter PEG chains. The choice of cross-linker also had profound effect on the hardness

42

4 RESULTS AND DISCUSSION

600110016002100260031003600 (cm-1)

ASW

DIW

Fresh

Figure 4.4 FT-IR of hydrogel coating H2, freshly prepared (bottom), after 42 days in DIW (center), and after 42 days in ASW (top).

-10%

90%

190%

290%

390%

490%

0 5 10 15 20 25

H 1H 2H 3H 4H 5H 6H 7H 8

-10%

10%

30%

50%

70%

90%

0 5 10 15 20 25

Wei

ght d

iffer

ence

Days of immersion

-10%

190%

390%

590%

790%

990%

0 5 10 15 20 25

H 1H 2H 3H 4H 5H 6H 7H 8

-10%

10%

30%

50%

70%

90%

110%

0 5 10 15 20 25

Wei

ght d

iffer

ence

Days of immersion

Figure 4.2 Results from the swelling experiments in DIW with an inset of the lower region of swelling.

Figure 4.3 Results from the swelling experiments in ASW with an inset of the lower region of swelling.

43

4 RESULTS AND DISCUSSION

and the flexible TMP-PEG cross-linker yielded softer coatings in comparison to the triazine cross-linker. Interestingly, the presence of esters had a softening effect on the hardness and the purely ether based, thiol-allyl ether proved to be hardest.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

H1 H2 H3 H4 H5 H6 H7 H8 EP

Figure 4.5 Indentation hardness measurements on the hydrogel coatings as well as the primer.

0

20

40

60

80

100

H1 H2 H3 H4 H5 H6 H7 H8 EP

Dry

Swelled

Figure 4.6 Pendulum hardness measurements on the hydrogel coatings as well as the primer.

Another important parameter for a marine coating is its adhesion to the underlying substrate or primer. This parameter is often neglected when designing a novel antifouling strategy127. However, without a good adhesion to the layer beneath, the antifouling properties of the top coat cannot be fully utilized. In order to evaluate the adhesion of the hydrogel top coats, crosshatch adhesion measurements were performed in both the dry and the swelled state (Table 4.1). It was found that all systems, in general, had good adhesion to the underlying primer in both the dry as well as the swelled state.

44

4 RESULTS AND DISCUSSION

4.1.6 In vitro antifouling studies

As stated in the introduction, the fouling process is considered to start with protein adsorption followed by colonization of microorganisms such as bacteria and diatoms, and finally colonization of macroorganisms. Hence, initial antifouling properties were evaluated in vitro, using proteins, bacteria and diatoms.

Protein adsorption, which is considered to be the first stage of fouling, was evaluated using FITC-labeled BSA (Figure 4.7). It was found that most systems had protein resistant properties; however, the allylic coatings containing esters (H5 and H6) had quite poor protein resistance. Another conclusion was that the length of the PEG chain affects the protein resistance, i.e. longer PEGs provided improved performance compared to their shorter analogues.

0,00E+00

5,00E+06

1,00E+07

1,50E+07

2,00E+07

2,50E+07

3,00E+07

Tota

lfluo

resc

ence

(A.U

.)

Figure 4.7 Results from the protein adsorption studies using FITC-labeled BSA in PBS solution.

The second stage of fouling, as described in chapter 2.4.1.1, is the colonization by bacteria and other microorganisms such as diatoms. Accordingly, one type of marine bacteria, Cobetia marina, and one type of diatom, Amphora coffeaeformis, were used for in vitro antifouling tests. In the case of the marine bacteria, a bioassay study evaluating its formation, as well as the fouling release properties were performed (Figure 4.8). Most coatings inhibited bacterial growth and again the longer PEGs provided enhanced performance. Coatings H3 and H4, containing the flexible TMP-PEG cross-linker (6), provided the best growth inhibition whereas the smooth allylic coating H5 provided the best fouling release properties. The settlement and formation of the diatom was also evaluated in a bioassay study (Figure 4.9). This study showed that most coatings inhibited the formation compared to the hydrophobic reference (Silastic T2); however, only coatings H3 and H4 provided better inhibition than the hydrophilic reference. In good accordance with the protein adsorption and bacterial bioassay, the results from the diatom assay showed that longer PEG chains provide enhanced antifouling properties.

45

4 RESULTS AND DISCUSSION

4.1.7 Field tests of hydrogel coatings

As the results from the in vitro antifouling tests will only show if the coatings are resistant to a specific molecule or organism, at the conditions in which the tests are performed, field tests are required to fully evaluate an antifouling coating. Accordingly, a field test in the North Sea harbor of Norderney, Germany was conducted in between the 27th of May to the 16th of September 2009 (Figure 4.10). The test clearly showed that the non-toxic hydrogel coatings were inferior to the modern biocidal coatings. However, when the hydrogel coatings are compared internally interesting differences can be seen. The coatings based on the longer PEG and triazine cross-linkers, H2 and H8 performed best. Interestingly, the purely ether based coating H8 provided somewhat better antifouling, possibly due to better hydrolytical stability compared to H2. Additionally, coatings comprising of the flexible TMP-PEG (6) cross-linker displayed deviant behavior compared to the other coatings.

0

500

1000

1500

2000

2500

3000

H1 H2 H3 H4 H5 H6Glass Silastic T2

FormationRelease

Coating

RFU

(Rel

ativ

e Fl

uore

scen

ce U

nit)

0

200000

400000

600000

800000

1000000

1200000

H1 H2 H3 H4 H5 H6Glass Silastic T2

Settlement5 days formation

Coating

RFU

(Rel

ativ

e Fl

uore

scen

ce U

nit)

Figure 4.8 Results from the bioassay study with Cobetia marina.

Figure 4.9 Results from the bioassay study with Amphora coffeaeformis.

46

4 RESULTS AND DISCUSSION

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Foul

ing

rate

Time of exposure (days)

H1

H2

H3

H4

H5

H6

H7

H8

EP

Figure 4.10 Fouling rate of the hydrogel coatings and the primer.

4.2 Synthesis of linear dendritc hybrids and their potential applications

Using macromolecular architecture as a tool for the design of novel functional materials is an area that has become very popular. Advances in synthetic methodologies during the last decades have allowed polymer chemists to afford a wide range of polymers with controlled molecular weights and end-group functionalities, including dendritic polymers. This has also allowed for the emergence of a new family of polymers, the linear dendritic hybrids, allowing for the design of novel macromolecular architectures. As many of the contemplated applications of these materials are within the biomedical field, we have chosen building blocks known for their biocompatibility and non-toxic nature. Using bis-MPA dendrons as our dendritic part, a library of materials have been synthesized. By choosing two different linear polymers, PEG and PCL, having different polarities (PEG hydrophilic and PCL hydrophobic) amphiphillic materials were afforded. These materials were subsequently evaluated as candidates for the formation of drug delivery applications (4.2.2.1) and isoporous membranes (4.2.2.2).

47

4 RESULTS AND DISCUSSION

4.2.1 Synthesis of linear dendritic hybrid block-copolymers using ROP and click chemistry

Using dendrons, of generation 0 to 4, bearing a click functionality (in this case an alkyne or allyl) in the core and peripheral hydroxyl groups as macroinitiators for ROP of ε-CL, a library of star branched polymers of DLn type were synthesized (Scheme 4.6). The library was initially designed with PCL arms of DP 30 and DP 60 on each generation of the dendrons bearing the alkyne core functionality. This results in an overlap in molecular weight in between every generation step, e.g. alk-G1-PCL60 has a similar molecular weight as alk-G2-PCL30. In addition, a few selected materials bearing allyl functionality in the core were synthesized. DSC measurements on these materials revealed no differences due to change in generation, except from G0 to G1 where melt and crystallization temperatures and enthalpies were slightly lowered comparing G1 to G0 (Table 4.3).

Scheme 4.6 General procedure for the synthesis of linear dendritc hybrid materials using ROP and click chemistry.

X O Gn O H X OGn O O H

On

YX OGn O O H

On O

m

O

O

O Ym

N NN **

*S *

Sn(O c t) 2T oluene, 110°C

C uAAC orT hio l-ene

X = or Y = N 3 or SH YX = or

In order to render our library of materials amphiphilic, azide functional PEGs (2000 Da and 5000 Da) were attached using CuAAC click chemistry yielding LDLn structures (Figure 4.11). A slight excess (1.5 equivalents) of the azide functional PEGs was required to achieve full conversion of the alkynes. It was found that the higher generations (G3 and G4) of the star branched polymers required higher reaction temperatures (60°C) and longer reaction times (4-5 days) compared to the lower generations (G0-G2), which were completed within 24-72 h at 40 °C. The excess of PEG could easily be removed by precipitation in methanol. As an illustrative example, 1H-NMR and SEC of the PEG2k-G1-PCL60 is shown together with its starting materials (Figure 4.12 and Figure 4.13). The conversion of the alkyne can readily be followed as the doublet at 4.7 ppm (g) is converted into a singlet at 5.25 ppm. The incorporation of the PEG chain can also be seen by the broad peaks from the PEG backbone at 3.6 ppm (b and c), thus demonstrating that the targeted material was afforded. The SEC (Figure 4.13) show the successful purification, removing the excess PEG. However, the amphiphilic product is eluted slightly later than the star branched precursor and with a slightly broader peak which

48

4 RESULTS AND DISCUSSION

could indicate that some degradation has occurred. Nevertheless, 1H-NMR shows that the product was formed and the PDIs were still low (≤1.2). As the materials are targeted for biomedical applications residual copper may be of some concern. Thus a simple purification protocol, involving precipitation of the products in a methanol:water mixture containing 1% EDTA, was developed and successfully utilized. This enabled us to turn the slightly green products into white products; although, further studies are required in order to determine the efficiency of this method.

Figure 4.11 Structures of hybrid materials PEG-Gn-PCL, from generation 0 to 4.

ON

n

N NO

OH

O

n O

Nn

N N O

OO

O

O H

O

H

O

O

n

n

ON

n

N N

OO

O

O

O

H

O

H

O

O

n

n

OO

O

O

O

O

O

H

O

H

O

On

n

ON

n

N N

O O

OO

O

O

OO

O

O

O

H

OH

O

H

OO

O

OH

O

n

n

n

n

O O

OO

O

O

OO

O

O

O

O

O

H

OH

O

H

OO

O

OH

O

n

n

n

n

ON

n

N N

OO

OO

OO

O O

OO

O

O

O

O

O

O

O

O

O

O

O

OO

H

O

O HO

O

H

O

O

H

O

HO

H

O

H

O

H

O

O

OO

O

n

n

n

n

n

n n

n

OO

OO

OO

O O

OO

O

O

O

O

O

O

O

O

OO

O

O

O

O OH

O

O HO

O

H

O

O

H

O

HO

H

O

H

O

H

O

O

OO

O

n

n

n

n

n

n n

n

PEG-G0-PCL PEG-G1-PCL PEG-G2-PCL

PEG-G3-PCL

PEG-G4-PCL

49

4 RESULTS AND DISCUSSION

OO

N 3n

O

OO HO

On

O

OO HO

O

n

O

O

OO

Nn

N N

O

OO HO

On

O

OO HO

O

n

O

O

1.52.02.53.03.54.04.55.05.56.06.57.07.58.0 (ppm)

a b

c d

e f g

h

ij

k

l

m

n

a

b, c

defg

h

i

j k, m l

aa

b

c

b, c

d

d

e

e

g

g

h

h

i

i

j

k

j l

m

n

n’

k, m ln

n

n’

Figure 4.12 1H-NMR of alk-G1-PCL60, PEG2k-N3 and the product PEG2k-G1-

PCL60.

Figure 4.13 SEC traces of PEG2k-N3, alk-G1-PCL60 and PEG2k-G1-PCL60.

14 15 16 17 18 19 20

PEG2k-G1-PCL60

alk-G1-PCL60

PEG2k-N3

By using thiol-ene chemistry the use of metal catalysts can be circumvented. Thus, a few selected materials, including TE-PEG2k-G1-PCL60 and TE- PEG2k-G4-PCL30 were synthesized. The reactions proved to be fast, and by using 3 equivalents of thiol functional PEG, the amphiphilic linear dendritic hybrids could be afforded within 30 min. Again, 1H-NMR (Figure 4.14) was used to follow the reaction and the shift of the peaks f and h together with the

50

4 RESULTS AND DISCUSSION

14 15 16 17 18 19

TE-PEG2k-G1-PCL60

all-G1-PCL60

PEG2k-SH

Figure 4.15 SEC traces of PEG2k-N3, alk-G1-PCL60 and PEG2k-G1-PCL60.

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

ab

c

d

e

f

g

h

i

jk

l

m

n

ab

c

d

e f

g

h

i

jk

l

m

n

OOn

O

S H

OOn

O

SO

OO HO

On

O

OO HO

O

n

O

O

O

OO HO

On

O

OO HO

O

n

O

O

o

o

o’

o’

o l, n mk

j i

hg f

a

b, c

c’ d, eSH

c’, h, j

o b, c

a

d, e, f

k, n m

i

Figure 4.14 1H-NMR of alk-G1-PCL60, PEG2k-N3 and the product PEG2k-G1-

PCL60.

peaks b and c from the PEG confirmed the successful synthesis. SEC (Figure 4.15) further showed successful synthesis and purification, this time the product eluated at shorter eluation times and without broadening in difference to the CuAAC route.

51

4 RESULTS AND DISCUSSION

4.2.2 Applications of the amphiphilic linear dendritic hybrids

As our hybrid materials were designed to afford amphiphilic properties they are expected to self-assemble if put under the appropriate conditions. This could enable the materials to create micelles or isoporous membranes, hence, the ability of our materials to self-organize into these types of structures were evaluated.

4.2.2.1 Formation of nanosized micellesIn our studies it was found that materials having a weight percent PCL

higher than 80% were unable to form micelles stable enough to allow for further characterization. Thus, four of our materials synthesized using CuAAC click chemistry qualified for further evaluation: PEG2k-G0-PCL60, PEG2k-G1-PCL30, PEG5k-G1-PCL60, and PEG5k-G2-PCL30 (Table 4.2). These materials can be divided into two pairs, PEG2k-G0-PCL60 and PEG2k-G1-PCL30, and PEG5k-G1-PCL60, and PEG5k-G2-PCL30. As both parts of each pair have similar molecular weight the effect of the dendron can be analyzed. Interestingly, increasing the generation has effect on both the CMC as well as the size of the micelles. The CMC is lowered and the size of the micelles is increased by increasing the generation. This could be seen as evidence of linear dendritic materials of a higher generation with shorter arms cannot pack themselves as closely as the one of a lower generation with longer arms. Finally, the micelles were loaded with the potent anti-tumor drug Doxorubicin. It was found that most micelles (materials based on G0 and G1) displayed good loading efficiencies (16-18 wt%); however, the micelles based on the G2 hybrid material had only 7 wt% loading efficiency. Again this could be seen as evidence of a lower degree of packing of the hydrophobic PCL in the core of the micelle.

Material Ratio PCL/PEGtheo

CMCa (μg∙ml-1)

dDLS, Intb

(nm)dDLS, Vol

b (nm)

dDLS, Numb

(nm)PDIb Loading

eff. (wt%)

PEG2k-G0-PCL60 3.4 98 ± 2 36 ± 3 21 ± 3 21 ± 3 0.26 ± 0.01 18

PEG2k-G1-PCL30 3.4 133 ± 2 77 ± 3 31 ± 3 31 ± 3 0.17 ± 0.02 17

PEG5k-G1-PCL60 2.7 113 ± 7 50 ± 2 33 ± 2 33 ± 2 0.27 ± 0.01 16

PEG5k-G2-PCL30 2.7 79 ± 3 54 ± 2 41 ± 2 41 + 2 0.15 ± 0.01 7a Determined by the fluorescent probe technique. b Determined by DLS at a concentration of 0.34 mg∙ml-1.

Table 4.2 Characterization of micelles.

52

4 RESULTS AND DISCUSSION

Mat

erial

Mn,

theo

(g

∙mol

-1)

Mn,

NM

R,

core

(g∙m

ol-1)

Mn,

NM

R,

end-

grou

p (g

mol

-1)

Mn,

SECCC

(g

∙mol

-1)

PDI,

SECCC

Mw,

SE

CTD

(g∙m

ol-1)

Rg,

SECTD

(nm

)

PCL,

th

eo w

t%T m

(C

º)ΔH

m(J∙

g-1)

T c (Cº)

ΔHc

(J∙g-1

)St

able

mice

llePo

rous

fil

m

alk-G

0-PC

L30

3481

4400

4200

6800

1.12

4800

2.7

9852

.878

.731

.877

.7

alk-G

0-PC

L60

6905

6700

7600

1070

01.

1078

003.

499

54.4

77.4

32.7

79.2

alk-G

0-PC

L240

2745

241

600

1890

026

400

1.21

2060

05.

8>

99

alk-G

1-PC

L30

7021

7200

8100

1150

01.

1381

003.

698

51.3

67.3

29.7

67.8

alk-G

1-PC

L60

1387

012

400

1050

022

800

1.15

1640

05.

299

54.4

64.3

30.8

64.7

alk-G

1-PC

L120

2756

823

500

3060

031

300

1.13

2120

06.

299

alk-G

2-PC

L30

1410

216

800

1790

021

900

1.16

1950

05.

097

52.5

66.6

25.6

67.3

alk-G

2-PC

L60

2780

028

900

3370

041

100

1.13

3070

07.

199

54.8

65.4

29.8

66.4

alk-G

3-PC

L30

2826

532

700

3530

039

300

1.11

3890

06.

897

52.3

66.0

28.5

68.0

alk-G

3-PC

L60

5566

148

400

7030

062

300

1.05

6510

08.

898

53.7

63.5

26.7

63.5

alk-G

4-PC

L15

2919

425

000

3470

036

300

1.06

4470

06.

394

alk-G

4-PC

L30

5659

058

900

7010

058

300

1.06

6300

08.

297

51.7

67.5

27.2

68.0

all-G

1-PC

L60

1387

214

700

1560

023

000

1.11

1520

05.

299

53.9

73.2

30.4

73.5

all-G

3-PC

L30

2826

732

400

3590

041

100

1.07

3960

06.

797

all-G

4-PC

L30

5659

234

500

6070

050

900

1.05

6370

07.

697

50.9

72.3

21.4

72.4

PEG

2k-N

320

2427

001.

0552

.215

5.8

23.6

150.

5

PEG

5k-N

350

2465

001.

1158

.816

1.8

33.7

158.

5

PEG

2k-S

H20

8828

001.

0551

.514

8.9

29.7

148.

5

PEG

2k-G

0-PC

L60

8905

1190

01.

0712

000

4.0

7750

.882

.628

.589

.0Y

PEG

2k-G

0-PC

L240

2945

226

700

1.19

2070

06.

093

Tabl

e 4.

x. P

rope

rties

of

linea

r den

driti

c hy

brid

s and

thier

pre

curs

ors.

53

4 RESULTS AND DISCUSSION

Mat

erial

Mn,

theo

(g

∙mol

-1)

Mn,

NM

R,

core

(g∙m

ol-1)

Mn,

NM

R,

end-

grou

p (g

mol

-1)

Mn,

SECCC

(g

∙mol

-1)

PDI,

SECCC

Mw,

SE

CTD

(g∙m

ol-1)

Rg,

SECTD

(nm

)

PCL,

th

eo w

t%T m

(C

º)ΔH

m(J∙

g-1)

T c (Cº)

ΔHc

(J∙g-1

)St

able

mice

llePo

rous

fil

m

PEG

2k-G

1-PC

L30

9021

1240

01.

1012

600

4.0

7649

.655

.027

.051

.3Y

PEG

2k-G

1-PC

L60

1587

019

700

1.20

1920

05.

486

53.8

59.9

28.6

60.8

Ireg

PEG

2k-G

1-PC

L120

2956

830

800

1.12

2670

06.

893

PEG

5k-G

1-PC

L60

1887

019

200

1.16

2230

05.

973

54.5

74.5

29.7

71.7

Y

PEG

2k-G

2-PC

L30

1610

218

900

1.22

2270

05.

385

51.8

58.1

26.7

60.4

Iso

PEG

2k-G

2-PC

L60

2980

036

400

1.12

3000

06.

792

54.5

59.9

27.8

60.1

Iso

PEG

5k-G

2-PC

L30

1910

219

200

1.21

2600

05.

672

50.6

54.5

23.8

53.3

Y

PEG

5k-G

2-PC

L60

3280

030

700

1.20

3870

07.

484

53.8

50.1

30.9

54.7

PEG

2k-G

3-PC

L30

3026

534

500

1.10

4390

07.

091

52.9

58.3

31.7

57.7

Iso

PEG

2k-G

3-PC

L60

5766

153

900

1.06

6480

08.

895

53.1

62.0

24.6

62.5

Iso

PEG

5k-G

3-PC

L30

3326

533

900

1.15

5060

07.

482

51.4

61.6

25.6

57.3

PEG

5k-G

3-PC

L60

6066

143

300

1.17

6480

08.

890

53.5

62.7

27.1

63.2

PEG

2k-G

4-PC

L15

3119

434

000

1.07

4780

06.

388

PEG

2k-G

4-PC

L30

5859

050

200

1.08

7830

08.

394

51.7

64.2

24.9

65.0

Ireg

PEG

5k-G

4-PC

L30

6159

036

700

1.14

7400

08.

389

51.4

55.4

27.9

58.3

TE-P

EG

2k-G

1-PC

L60

1596

023

600

1.16

8652

.959

.929

.960

.7

TE-P

EG

2k-G

3-PC

L30

3026

738

000

1.07

5030

07.

591

TE-P

EG

2k-G

4-PC

L30

5868

046

700

1.05

9350

.565

.025

.365

.8CC

Det

erm

ined

usin

g co

nven

tiona

l cali

brat

ion.

TD D

eter

min

ed u

sing

tripl

e de

tect

ion

calib

ratio

n.

Tabl

e 4.

x. C

ontin

ued

54

4 RESULTS AND DISCUSSION

4.2.2.2 Formation of ordered honeycomb membranesIn order to study the self-organization of the amphiphilic hybrid materials

into isoporous membranes, the materials were cast from benzene onto a glass slide in a humid environment. Initially, amphiphiles with varying DP of the PCL chain (DP 30 or 60), generation (G0-G4), and constant PEG length (PEG2k), were cast and their resulting films were studied with optical microscopy (Figure 4.16). It was found that porous films were formed for the all but two of the materials, PEG2k-G0-PCL60 and PEG2k-G1-PCL30. However, out of these six materials four could be considered isoporous, PEG2k-G2-PCL30, PEG2k-G2-PCL60, PEG2k-G3-PCL30 and PEG2k-G4-PCL60. Interestingly, the effect of the dendron is clearly seen, as even though PEG2k-G1-PCL60 and PEG2k-G2-PCL30 have the same molecular weight, only the G2 forms an isoporous structure. A similar effect can be seen when comparing PEG2k-G3-PCL60 to PEG2k-G4-PCL30. The film of the PEG2k-G3-PCL30 proved to produce the most perfect membranes and was further studied using SEM. It could be observed that the pores were formed as a thin monolayer with a thickness of about 1 μm. Furthermore, the pores had a diameter of about 3 μm and a wall thickness of about 0.2 μm.

To further study the effect of the dendron additional materials were synthesized all having the same molecular weight and hydrophobic/hydrophilic ratio but varying the generation from G0 to G4. Since PEG2k-G3-PCL30 was considered to produce the best honeycomb membranes, this was the targeted molecular weight for the additional synthesis. Hence, PEG2k-G0-PCL240, PEG2k-G1-PCL120 and PEG2k-G4-PCL15 were synthesized in order to allow an elucidating study. It was found that materials based on generations two and three produced isoporous structures whereas materials based on generation zero and one produced irregular pores and the material based on generation four was unable to produce an isoporous structure. The results agree well the earlier observations that branched structures forms isoporous structures more readily105. Furthermore, earlier work on linear dendritic hybrids of (LD)n structures has also found that materials based on generation two and three provides the best materials111.

55

4 RESULTS AND DISCUSSION

PEG2k-Gn-PCL60 PEG2k-Gn-PCL30

Figure 4.16 Optical microscopy images of casted PEG2k-Gn-PCL60 (left) and PEG2k-Gn-PCL30 (right). PEG2k-G0-PCL60 (A), PEG2k-G1-PCL60 (B), PEG2k-G2-PCL60 (C), PEG2k-G3-PCL60 (D), PEG2k-G1-PCL30 (E), PEG2k-G2-PCL30 (F), PEG2k-G3-PCL30 (G), PEG2k-G4-PCL (H).

56

4 RESULTS AND DISCUSSION

Figure 4.17 SEM images of the top (left) and cross-section (right) on an isoporous surface fabricated from PEG-2K-G3-PCL30.

Figure 4.18 Optical microscopy images of casted PEG2k-Gn-PCL. PEG2k-G0-PCL240 (A), PEG2k-G1-PCL120 (B), PEG2k-G2-PCL60 (C), PEG2k-G3-PCL30 (D), PEG2k-G4-PCL15 (E).

57

5 CONCLUSIONS

5 CONCLUSIONS

To explore the use of stealthy PEG based hydrogels as marine antifouling coatings, a library of hydrogel networks was synthesized using thiol-ene chemistry. Parameters such as the length of PEG, cross-linking chemistry, chemical stability and cross-linker were varied. It was found that the curing chemistry had a large impact of the final materials. Thiol-methacrylate systems displayed a significant amount of homopolymerizations, whereas thiol-allyl ether systems cured more purely with thiol-ene coupling, as observed using FT-Raman. The presence of homopolymerization was further supported by the DSC measurements as the Tg was higher for the thiol-methacrylate systems compared to the thiol-allyl ether systems. Furthermore, DSC revealed that the presence of hydrolytically labile esters also had a profound effect on the thermal behavior of the systems. Swelling and degradation studies showed that the stability of the coatings was reduced for coatings containing esters compared to purely ether based coatings. To evaluate the antifouling properties, in vitro tests using with a protein, a marine bacteria and a diatom were initially performed. The tests showed that, coatings comprising of PEGs having a higher molecular weight performed better than coatings comprising of short PEGs. It was also found that the choice of cross-linker affected the performance. The performance of the coatings was further analyzed in a field test as well as characterization of its mechanical properties. The field test displayed that although the results from the in vitro studies were promising, none of the coatings could compete with modern biocidal antifouling coatings. However, as in the in vitro studies, the coatings comprising of longer PEG chains displayed better performance compared to the coatings comprising of short PEGs. Furthermore, the hydrolytically stable coatings, purely based on ethers, performed better than coatings containing esters. Additionally, the choice of cross-linker affected the antifouling behavior. Finally, the mechanical properties of the coatings were characterized and, as expected, longer PEGs and swelling of the coatings resulted in softer coatings. In addition, the adhesion of the coatings to primed PVC panels was evaluated showing that all coatings adhered both in the dry and swelled state.

Other applications of stealthy materials are within the biomedical field such as drug delivery systems and sensors. Accordingly, a library of linear dendritic hybrid materials, with intrinsic biocompatibility, was synthesized using ROP and click chemistry. Using dendrons bearing a click functionality in the core (either alkyne or allyl) and peripheral hydroxyl groups as a macroinitiator, a library of star branched materials was afforded with dendrons from G0-G4 and varying PCL length. These materials were subsequently reacted with a PEG

58

5 CONCLUSIONS

(2000 Da or 5000 Da), bearing the opposing click functionality (azide or thiol), yielding the final amphiphillic linear dendritic hybrids of LDLn type. It was found that both types of click chemistry, CuAAC and thiol-ene, resulted in the targeted materials. Thiol-ene chemistry resulted in the targeted materials faster and without any evidence of degradation compared to the CuAAC chemistry, although thiol-ene chemistry required a higher excess of PEG, in order to afford complete conversion of allyl. Furthermore, the library of materials was used to make micelles. It was found that materials with a weight percentage of PCL above 80% did not yield stabile micelles. Additionally, it was found that the dendron affected the CMC as well as the size of the micelle as increasing the generation while maintaining the same molecular weight resulted in lower CMC values and larger micelles. The amphiphilic hybrid materials ability to form isoporous membranes was subsequently analyzed and it was found that the generation of the dendron had a profound impact. Dendrons of the 2nd and 3rd generation afforded isoporous structures, whereas materials based on G0, G1 and G4 were in some cases able to form pores, although, not of isoporous structure. Furthermore, it was found that materials comprising of the longer PEG (PEG5k) did not yield any isoporous structures.

59

6 FUTURE WORK

6 FUTURE WORK

PEG-based hydrogel materials for marine antifouling have a lot of potential. However, significant efforts are required to evaluate if this types of coatings can be competitive with the modern antifouling coatings. Further work should include increased length of the PEG chains and a more extensive variation of crosslinkers. Other interesting approaches could include the introduction of PEGs only attached in one end, hence affording both the benefits of a hydrogel and a SAM, or introduction of free PEG of very high molecular weight that could be released slowly with time. Finally, in order to make these types of coatings suitable for commercial use, the coating systems should be formulated so that they can cure at a suitable rate without the need for UV-curing. Furthermore, as PEGs are water soluble, a commercial formulation could be made with water as the only solvent, thus reducing the exposure to malicious solvents for the final user.

The linear dendritic hybrid materials afford almost unlimited variations enabling them to be tailored for a range of applications. With respect to the drug delivery applications further work should include more extensive drug loading and release studies as well as their effect on the targeted cells. For drug delivery it is expected that a longer PEG segment would produce more stable micelles which would enable a wider range of materials to be able to form micelles. Furthermore, by changing the place of PEG and PCL a whole new set of materials could be accomplished which should form more stable micelles. Further work should also include further functionalization of the end-groups, which could have a lot of potential both in the drug delivery case (e.g. targeting moieties) as well as for the isoporous membranes (e.g. cross-linking or molecules for sensor applications). Furthermore, there are a range of other possibilities for the isoporous membranes. Other polymers, such as non-crystalline polycarbonates should be compared to the crystalline PCL for the formation of isoporous membranes. Finally, the ultimate challenge is to fabricate a material that could be of commercial interest, e.g. a sensor material.

61

7 ACKNOWLEDGEMENTS

7 ACKNOWLEDGEMENTS

First of all, I would like to thank Professor Anders Hult for your support and for giving me the opportunity to pursue my PhD in the division of coating technology.

I would also like to thank Associate Professor Michael Malkoch for supervising me, and always taking time to help and guide me during these years.

Professor Mats Johansson is thanked for always taking time to help me, for coauthoring the hydrogel papers and for being a great Santa Claus.

Professor Eva Malmström is acknowledged for all her hard work in both the division, as well as deputy President at KTH. I don’t know how you can do it all and still find time for our interesting scientific and private discussions.

Assistant Professor Anna Carlmark Malkoch is thanked for being so nice and for taking over the polymer lecture at Forskarskolan after Eva.

Furthermore, the coauthors and other members for the antifouling project Cértol: Claes Lundin, Andreas Trygg Bigner, and Christian Bigner at Lotréc AB, Job Klijnstra, Glen Donnelly, and Anouk Bruin at TNO, Burkard Watermann, and Bernd Daehne at LimnoMar, and Professor Jerzy Falandysz at University of Gdansk.

Assistant Professor Andreas Nyström at the Swedish Medical Nanoscience Center at Karolinska Institutet is acknowledged for his assistance with practical and scientific guidance, and for coauthoring two of the included manuscripts. The other members of the nanoparticles for drug delivery targeted for cancer treatment project, Professor Dan Grander, and Theocharis Panaretakis at the Department of Experimental Onkology at Karolinska Institutet are also acknowledged.

I would also like to thank Sonny Jönsson, for your advice regarding UV-curing.

For financial support, the European Union’s Sixth Framework Program, project number COOP-CT-2006-032333, and the Swedish Research Council (VR), project number 2009-3259, are greatly acknowledged.

All friends, colleagues and members of “ytgruppen” are greatly acknowledged. Robert, thank you for all the fun times at work, for being part of “matlaget”, for all the fun trips and conferences, and for all your support. You are a true friend. Axel, is thanked for bringing the fondue pot, for being able to smell “fika” all the way from Flemmingsberg and for telling Hanna N. when she is cheating in Bang. Pelle is thanked for being “Doktorn” and for having the wettest wedding ever. Daniel N. is thanked for fun beach volley sessions, after works and all our Wang-experiments. Linda is thanked for drinking me under

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the table during my first year in “ytgruppen”, as I recall it usually ended with interesting discussions. Hanna L. is tanked for having a real “vinnarskalle”. Kattis is thanked for being a great ”bollplank” and for all the fun times. Camilla is thanked for always being so nice, Emma Ö. for being a great roomy and Fina for calling me “ytgruppens bäbis”. Mange is thanked for being a predecessor in the field of “sprängsmörjning”. Susanne is thanked for being dirty, even when you’re trying not to, and for going in my footsteps. Take care of both the SEC and Forskarskolan. Yvonne is thanked for being so “hurtig” and telling about the worst neighbor in history. Kim is thanked for being the second half of the short beach volley team and for his ability to sleep in the corner of my couch. Christian is thanked for being a kicker but doing the right thing and for meeting the dad on the first night. Linn is thanked for being so cheeky and for preferring me instead of Susanne. Carl is thanked for being a great chef and Marcus for being my Nemisis. Marie is thanked for great cooperation in the lab and for always listening to my ideas. Maribel, thank you for all the laughs in the lab. Mauro and Alireza are thanked for taking care of the IR and Stacy for learning Swedish so fast. Ting, I will always remember when you laughed at Christians hair. All my past and present roomies, Petra, Samira, Melissa, Suba, Hui, Lage, Kristina, Bella and Sandra, thank you for making this room, the best room. Inger is thanked for all the adiministrative help. Finally, I would also like to thank all other past and present members and visitors of “ytgruppen”, Emma L., Niklas, Jan, Sara K., Sara O., Martin R., Helena, Sandra B., Emelie, Daniel S., Jarmo, George, Neil, Kaname and everyone else that I have forgot to mention.

All the “Lunch Cowboys” are thanked for a great effort in Studentiaden. All other Professors and staff of the Department of Fibre and Polymer

technology for making this such a nice place to work. I would also like to thank all my friends outside KTH. Karlstadsgänget, Carl,

Jonas, Nils, Per, Christian and Henrik, thank you for all the good times. All the guys from ÖAAIS, thank you for the floorball, parties and cruises. I would also like to thank my former classmates in Uppsala.

Stort tack till Lena, Roger, Paulina, Martin och Niklas för att ni har välkomnat mig så varmt till familjen Eriksson.

Slutligen vill även tacka min familj som hela tiden stöttat och uppmuntrat mig. Utan er hade det aldrig gått. Mamma, Pappa, Marika, Robban, Rasmus och Jennifer. Ni är bäst!

Martina, du är det bästa som har hänt mig, tillsammans klarar vi allt. Jag älskar dig!

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