Aggregation behavior of a temperature- and pH-responsive ... · Aggregation behavior of a...

Aggregation behavior of a temperature- andpH-responsive diblock copolymer in aqueous solution

Micael Garrido Gouveia

Thesis to obtain the Master of Science Degree in

Bioengineering and Nanosystems

Supervisor(s): Prof. Dr. Christine Maria Papadakis andProf. Dr. José Paulo Sequeira Farinha

Examination Committee

Chairperson: Prof. Dr. Luı́s Joaquim Pina da FonsecaSupervisor: Prof. Dr. Christine Maria Papadakis

Members of the Committe: Prof. Dr. José Manuel Gaspar Martinho

June 2016

What I cannot create, I do not understand.Richard Feynman

Acknowledgments

As opening courtesy, I would like to express my heartfelt gratitude to my supervisor Prof. Dr.

Christine Papadakis who accompanied and guided my work since day one, in every stage of this

research project, which she kindly accepted. Not only because she showed constant availability for

fruitful and encouraging weekly discussions, but also because of the great level of patient and will

power to help me to become a better scientist. As it is not enough, she gave me the opportunity to

participate in activities such as scientific workshops in Grainau and Aachen, from which I’ve learned

a lot and allowed me to explore the astonishing Germany as well. For that I will always be grateful.

On equal grounds, I would like to thank Prof. Dr. José Paulo Farinha for the suggestion for this

research project topic and for the opportunity to work in the CQFM group under supervising of Dr.

Carina Crucho who I thank for the time spent. I would like to acknowledge as well all people that I

contacted in during my work CQFM at that contributed for my learning and, of course for a cheerful

environment.

Furthermore, I wish to give my special thanks to Margarita Dyakonova, Bart-Jan Niebuur, Xiaohan

Zhang and Natalya Vishnevetskaya, my office comrades that in one way or another guided me through

my work, in a way they all were my unofficial supervisors. In almost every issue and problem or even

just discussing topics that I was unclear of, they truly and readily made time to help me. And of course,

thank you for the all nice and silly conversations that made the office a nice place to be and work.

Also, one great share of gratitude goes to all people of the E13 group that are the living proof that

a group can be fun, silly and productive at the same time. All the next office exploding laughter would

cheer me up any day, any time. However, I should express a special thanks to my dear friends Ali

Ozku, Richard Stockhausen, Edoardo Barabino and Chih-Chun Huang for turning the coffee time so

cheerful, and for making me feel at home.

Last but not least, a big thank you to my friends at home that kept in touch in a away that almost

was like I never left Portugal. But to my family, I am most grateful. I would like to thank my parents for

the unconditional love, support and for giving me the opportunity to go abroad and enjoy one in a life

time experience. For that, and much much more, thank you.

iii

Abstract

Orthogonally switchable diblock copolymers show rich phase behavior in aqueous solution, in

dependence on external stimuli. This behavior can be utilized for the preparation of ’smart’ drug de-

livery systems, which are tuned to respond in accordance to a specific physiological environment.

We investigate the aggregation behavior in water of a dual-stimuli responsive diblock copolymer with

a pH-responsive block composed by (diisopropylamino) ethyl methacrylate (DPA), a basic tertiary

amine-based monomer, and a temperature-responsive block composed by 2-(2-methoxyethoxy) ethyl

methacrylate (MEO2MA), a polyethylene glycol (PEG) analogue monomer, featuring a lower criti-

cal solution temperature (LCST). In the present work, the temperature- and pH-responsive PDPA-

b-PMEO2MA diblock copolymer was successfully synthesized by reversible addition-fragmentation

chain transfer (RAFT) polymerization, as confirmed by 1H NMR spectroscopy. The cloud-point tem-

perature (Tcp) was determined through turbidimetry for different pH values, and the polymer hydro-

dynamic radii dependence on pH at room temperature was evaluated by fluorescence correlation

spectroscopy (FCS), and near Tcp by temperature-resolved fluorescence correlation spectroscopy (T-

FCS). The characteristic length of the aggregates formed was also assessed below and above Tcp,

under acidic and alkaline conditions, by small-angle X-ray scattering (SAXS). Below Tcp, our exper-

iments have shown the formation of small aggregates with different densities depending on the pH

in solution, mainly attributed to the presence/absence of electrostatic repulsions arising from proto-

nated/deprotonated amine groups and to the increased hydrophilicity of the temperature-responsive

block under neutral and alkaline conditions. Additional large aggregates were formed above Tcp, which

was found to increase above acidic pH values.

Keywords

pH/temperature-responsive polymers, amphiphilic diblock copolymer, (diisopropyl amino)ethyl methacry-

late (DPA), 2-(2-methoxy ethoxy)ethyl methacrylate (MEO2MA), fluorescence correlation spectroscopy

(FCS), small-angle X-ray scattering (SAXS)

v

Resumo

Nesta tese foi investigado o mecanismo de agregação em água de um polı́mero de dibloco

sensı́vel a dois estı́mulos simultaneamente, com um bloco pH-sensı́vel composto por (diisopropil

amino) etil metacrilato (DPA), um monómero baseado numa amina terciária, e um bloco termos-

sensı́vel composto por 2-(2-metoxietoxi) etil methacrilato (MEO2MA), um monómero análogo ao poli-

etileno glicol (PEG), que exibe temperatura crı́tica em solução (LCST). O copolı́mero de dibloco

PDPA-b-PMEO2MA sensı́vel ao pH e à temperatura em simultâneo foi sintetizado por polimerização

de transferência reversı́vel de cadeia por adição-fragmentação (RAFT) e confirmado por análise de1H NMR. A temperatura do ponto de nuvem (Tcp) foi determinada através de turbidimetria, para difer-

entes valores de pH, enquanto a dependência do raio hidrodinâmico do polı́mero em função do pH foi

testado através de espectroscopia de correlação de fluorescência (FCS) à temperatura ambiente, e

para valores próximos à Tcp por espectroscopia de correlação de fluorescência de temperatura con-

trolada (T-FCS). A distância de correlação dos agregados foi também determinada para temperaturas

acima e abaixo da Tcp, em condições acı́dicas e alcalinas, por espalhamento de raio-X a baixo ângulo

(SAXS). Abaixo da Tcp, os resultados indicaram a formação de pequenos agregados que, consoante

o pH em solução, exibiram densidades distintas dependendo da presença/ausência de repulsóes

electrostáticas contralada pelo estado protonado/desprotonado dos grupos amina, e do aumento de

hidrofilicidade do bloco termossensı́vel em solução alcalina. Adicionalmente, verificou-se a formação

de agregados de maiores dimensões para temperaturas acima da Tcp, que por sua vez aumentou

para valores de pH superiores ao correspondente a condições acı́dicas.

Palavras Chave

polı́mero pH/termossensı́vel, copolı́mero de dibloco anfifı́lico, (diisopropil amino)etil metacrilato

(DPA), 2-(2-metoxietoxi) etil metacrilato (MEO2MA), espectroscopia de correlação de fluorescência

(FCS), espalhamento de raio-X a baixo ângulo (SAXS)

vii

Contents

1 Introduction 1

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2.1 Amphiphilic block copolymers: self-assembly . . . . . . . . . . . . . . . . . . . . 5

1.2.2 Temperature-responsive polymers . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2.2.A Collapse mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2.3 pH-responsive polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.3 Polymers under study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.4 Rationale and objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2 Methods 15

2.1 Controlled/living radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.1.1 RAFT principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.1.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.1.3 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.2.1 FCS setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.2.1.A FCS for micellization and aggregation studies . . . . . . . . . . . . . . 24

2.2.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.2.3 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2.2.3.A Triplet decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.2.3.B Multiple diffusion species . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.3 Small-Angle X-Ray Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.3.1 SAXS principle and setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.3.1.A SAXS setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2.3.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.3.3 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2.3.3.A Data Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3 Results and discussion 43

3.1 Characterization of the diblock copolymers after synthesis . . . . . . . . . . . . . . . . . 44

3.1.1 1H NMR spectra analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

ix

3.2 Diffusional studies of the self-assembled structures . . . . . . . . . . . . . . . . . . . . . 50

3.2.1 pH dependence of the aggregation behavior at room temperature . . . . . . . . 52

3.2.2 Temperature dependence of the aggregation behavior . . . . . . . . . . . . . . . 56

3.2.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3.3 Structure of the aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.3.1 Structure dependence on temperature and pH . . . . . . . . . . . . . . . . . . . 62

3.3.2 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

4 Conclusions and Final remarks 67

Bibliography 71

Appendix A Appendix A-1

x

List of Figures

1.1 Schematic representation of plasma concentration profile of therapeutic drugs for dif-

ferent delivery approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Schematic illustration of a diblock copolymer self-assembly and dissociation in re-

sponse to changes in pH or temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3 Formation of micelles in a selective solvent for one of the blocks and dependence of the

unimer and micelles concentration as function of the polymer concentration in solution. 6

1.5 Influence of the dimensionless packing parameter of polymer chains in the resulting

self-assembly structures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6 Schematic phase diagrams associated with LCST (lower critical solution temperature)

and UCST (upper critical solution temperature) behavior. . . . . . . . . . . . . . . . . . 8

1.7 Schematic of the cooperative hydration of LCST type temperature-responsive polymers. 9

1.8 Schematics of the effects of the solution pH in micelles composed by an hydrophobic

core and a polybasic pH-responsive shell. . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.10 Molecular structure of standard linear PEG and nonlinear PEG-analogs constructed

with poly(ethylene glycol) monomers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.12 Chemical structure of the PDPA-b-PMEO2MA diblock copolymer and the schematics

of the expected behavior in aqueous solution under temperature and pH stimuli. . . . . 13

2.1 Scheme for conventional radical polymerization. . . . . . . . . . . . . . . . . . . . . . . 16

2.3 Schematics for reversible deactivation and reversible chain transfer. . . . . . . . . . . . 17

2.4 Mechanism of RAFT polymerization and overall reaction. . . . . . . . . . . . . . . . . . 18

2.5 Mechanism of block copolymer synthesis through the RAFT technique. . . . . . . . . . 19

2.6 Schematic representation of a typical confocal FCS setup and its principle of operation. 23

2.7 Scheme of the chemical structure of Rhodamine 6G . . . . . . . . . . . . . . . . . . . . 24

2.8 Sketch of the temperature-resolved FCS frame setup . . . . . . . . . . . . . . . . . . . . 26

2.9 Photographs of the custom-made temperature-resolved FCS sample holder. . . . . . . 27

2.10 Custom-made system for gold deposition onto the edges of the ITO slips. . . . . . . . . 28

2.11 Autocorrelation curves for different particle numbers (N) and diffusion time (τD). . . . . . 30

2.12 Autocorrelation functions of two different species present in the detection volume with

varying relative amplitude (ρi) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.13 Scheme of the interaction of X-rays with matter. . . . . . . . . . . . . . . . . . . . . . . . 33

xi

2.14 Interference of detected scattered coherent waves and geometrical approach of point

scatter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2.15 Scheme of a SAXS instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.16 GANESHA 300XL SAXS system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.18 Schematics of the origin of parasitic scattering, and an example of sample masking

using Fit2D software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

2.19 SAXS capillary sample holder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2.20 The power law describing large aggregates for different parameters. . . . . . . . . . . . 40

2.21 The Ornsteins-Zernicke (OZ) contribution with different parameters describing density

fluctuations in small aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.2 Chemical structure of the of the DPA and MEO2MA monomers and of the temperature

and pH-responsive PDPA-b-PMEO2MA diblock copolymer. . . . . . . . . . . . . . . . . 45

3.6 1H NMR spectra used for the characterization of the DPA monomer and the polymer. . . 48

3.7 Normalized transmittance curves and photos of the cuvettes containing the samples

taken before and right after the turbidimetry measurements. . . . . . . . . . . . . . . . . 51

3.8 Normalized FCS autocorrelation functions from the samples with 0 mg/mL of polymer

concentration, i.e. only Rh6G in aqueous solutions, for pH 3, 6, 7 and 10, at room

temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

3.10 Normalized FCS autocorrelation functions of aqueous solutions of PDA-b-PMEO2MA

for concentrations ranging from 0.01 to 1.2 mg/mL and different pH values, at room

temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.11 Results from FCS measurements of PDPA-b-PMEO2MA aqueous solutions at various

pH 3, 6,7 and 10 at room temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.12 Temperature calibration of the temperature-resolved FCS system. . . . . . . . . . . . . 56

3.14 Normalized temperature-resolved FCS autocorrelation functions for different pH condi-

tions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

3.15 Results from temperature-resolved FCS measurements of a 0.8 mg/mL polymer solu-

tions at pH 3, 6, 7 and 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

3.18 SAXS scattering curves of aqueous solution with a concentration of 50 mg/mL at pH 6

(T=23°C and 28°C) and at pH 10 (T=23°C and 28°C) . . . . . . . . . . . . . . . . . . . 63

xii

List of Tables

3.1 Characteristics of DPA monomer synthesis and RAFT polymerization of the PDPA ho-

mopolymer and PDPA-b-PMEO2MA obtained from the PDPA as the macro-CTA . . . . 49

A.1 Hydrodynamic radii values from room temperature FCS measurements . . . . . . . . . A-2

A.2 Fraction of the slow decay values from room temperature FCS measurements. . . . . . A-2

A.3 SAXS model fitting parameters for pH = 6 sample . . . . . . . . . . . . . . . . . . . . . . A-3

A.4 SAXS model fitting parameters for pH = 10 sample . . . . . . . . . . . . . . . . . . . . . A-3

xiii

Glossary

AIBN 2,2-azobis(2-methyl-propionitrile) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

APD Avalanche photo diode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

CAC Critical aggregation concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

CMC Critical micelle concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

CMT Critical micelle temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

CRP Controlled/living radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

CTA Chain transfer agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

DDS Drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

DMF Dimethylformamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

DP Degree of polymerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

DPA 2-(diisopropylamino)ethyl methacrylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

FCS Fluorescence correlation spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

ITO Indium-tin-oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

LCST Lower critical solution temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

PEG Poly(ethylene glycol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

RAFT Reversible addition-fragmentation chain-transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

RTD Resistance temperature detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

SAXS Small angle x-ray scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

SDD Sample-to-detector distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

SLD Scattering length density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

TEA Triethylamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

TFA Trifluoroacetic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

THF Tetrahydrofuran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

UCST Upper critical solution temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

xv

List of Symbols

p Packing parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

a0 Surface area of the insoluble block at the interface soluble/insoluble blocks . . . . . 7

lc Length of the insoluble block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

v Volume of the insoluble block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Tcp Cloud-point temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Mn Number average molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

N Total number of fluorescent particles in the detection volume . . . . . . . . . . . . . 24

n Number of different fluorescent species . . . . . . . . . . . . . . . . . . . . . . . . 24

τD,i Diffusion time of the i th species . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

ρi Amplitude of the ith species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

z0 Half-height of the detection volume . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

w0 Half-width of the detection volume . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

TT Triplet fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

τT Triplet time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

τD,Rh6G Diffusion time of Rhodamine 6G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

DRh6G Diffusion coefficient of Rhodamine 6G . . . . . . . . . . . . . . . . . . . . . . . . . 25

δF (t) Fluorescence intensity variance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

F (t) Fluorescence intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

〈F (t)〉 Average fluorescence intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

G(τ) Normalized autocorrelation function . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

〈C〉 Average concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

V eff Effective detection volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

z0 Half-height of 3D Gaussian profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

w0 Half-width of 3D Gaussian profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

D Diffusion coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

rH Hydrodynamic radius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

kB Boltzmann’s constant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

T Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

η Solvent viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Ds Diffusion coefficient of the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

τD,s Diffusion time of the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

xvii

rH Hydrodynamic radius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

φ Azimuth angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

~q Scattering vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

q Momentum transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

bi Scattering length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

ρ Scattering length density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

ρ(~r) Scattering length density of the monomer . . . . . . . . . . . . . . . . . . . . . . . 37

ρs Scattering length density of the solvent . . . . . . . . . . . . . . . . . . . . . . . . . 37

I(~q) Scattered intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

f(~q) Scattering length amplitude . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

P (~q) Form factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

S(~q) Structure factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

δ Chemical shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

xviii

1Introduction

Contents1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.3 Polymers under study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.4 Rationale and objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1

1.1 Introduction

Over the paste few decades research activity in pharmaceuticals’ physico-chemical properties and

physiological impact have been increasing significantly, becoming a subject of academic and indus-

trial interest. With the emergence of novel drug therapies, there is an effort to redirect the attention

to the delivering approach, in an attempt to improve therapy efficiency. Drawbacks related with the

invasive character of intravenous drug administration (eg. infections) can be easily avoided by opt-

ing for oral route, being the preferred way of pharmaceuticals agents intake [1]. Factors related with

drug physico-chemical nature such as poor solubility, low membrane permeability and instability, as

well as poor pharmacokinetics hinder bioavailability for proper drug absorption [2]. Conventional drug

delivery systems (DDS) typically actuate in an uncontrollable way, leading to fluctuations of the local

and/or systemic drug concentration in the bloodstream, resulting in short windows of optimal drug

concentration, multiple drug administrations and potentially increase in its toxicity [3] (Figure 1.1).

Consequently, despite being in continuous development, conventional DDS are known to be still far

from featuring an ideal behavior, with unmet features concerning spatial and temporal release control.

Nanoscaled oral DDS have shown the ability to encapsulate a variety of drugs, which (i) avoids drug

degradation in the hostile environment of the gastrointestinal tract, (ii) enhances drug absorption by

facilitating diffusing through the intestinal membrane, and (iii) improves the pharmacokinetic release

profiles. Some of the current DDS are highlighted in the work of Mazzaferro et al. [4]. Additionally,

besides a good performance, a robust DDS is also required to be compatible to the human body,

which increases the need for the introduction of better and safer (bio)materials for encapsulation and

targeting purposes, which can provide a more efficient drug loading and release [5]. Consequently,

there is a need to investigate effective DDS, that allow therapeutic agents to be delivered in a timely

and localized manner, providing a transportation vehicle that improves drug bioavailability, efficiency

in drug absorption and ultimately patient compliance [6].

Figure 1.1: Schematic representation of plasma concentration profile of therapeutic drugs for different deliveryapproaches - conventional release of two drug administrations (orange line), controlled release (green line).MaEC- maximum effective concentration, MiEC-minimum effective concentration.

2

In order to tailor such pharmacological drugs in a safe and effective way, a compromise must

be maintained between bioavailability, toxicity and disposition within the body. In last years, various

modification techniques have been employed such as co-solvency, complexation, solid dispersion,

permeation enhancers, surfactants and others have been reported to help overcoming some solubil-

ity and permeability issues [7, 8]. Along possible therapeutic formulations, polymer colloidal systems

have been increasingly used in drug delivery field, as comprehensibly exposed in [9], in great part

due to the possibility of combining dynamic topological features of such systems and with the biocom-

patibility that some polymers present. These conditions may be met with polymers able to change

with its surrounding environment, in other words stimuli-responsive or smart-polymers, making pos-

sible the design of active and tailor-made therapeutic solutions, reviewed elsewhere [10].The interest

developed around stimuli-responsive polymers use is due to their ability to change abruptly theirs

properties upon an small physical/chemical stimulus - as defined by Hoffman [11] - depending on the

application, it could range from physical (temperature, magnetic field, light, etc), chemical (pH, spe-

cific ion etc) to biochemical (enzymatic substrates or affinity ligands such as glucose) [12, 13]. These

properties are given by the polymer nature, that could be synthetic, semi-natural or natural origin,

and by the fabrication method, permitting combination of stimuli by which polymers can be sensitive,

ideally resulting on a multifunctional system able to synchronize drug-release profiles with changing

physiological conditions [14, 15].

Temperature-responsive polymers are amongst the most widely investigated polymers due to their

potential biomedical applications and the easy to control stimulus. A temperature response can be

achieved via a number of mechanisms, including liquid crystal elastomer transitions [16], or solid

state transitions in shape memory materials [17, 18]. However, the solubility transition of the poly-

mer upon temperature change is the most commonly used response in drug delivery systems, as

well as in tissue engineering, gene delivery and others [19, 20]. In these cases, the transition tem-

perature of a polymer in solution is tuned to be within the biological range and can have an ex-

ternal (e.g. microwaves) [21] or internal origin (raise of body temperature upon disease) [19, 20].

Numerous polymers exhibit a temperature-responsive behavior, and they can be divided into two

major classes according to their origin: naturally occurring polymers and synthetic materials. Cel-

lulose, chitosan, xyloglucan, and gelatin, along with their derivatives, are some examples of nat-

ural temperature-responsive polymers. Synthetic materials such as poly(N-alkyl methacrylamide)s

are perhaps the most heavily studied temperature-responsive polymers, in particular, homopolymers

of the gold standard poly(N-isopropylacrylamide). However, due to its questionable biocompatibility,

other alternative polymers such as poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) [22], poly(2-

alkyl-2-oxazoline)s and poly(2-oxazines)s and poly(2-alkyl-2-oxazoline)s, and lactam/pyrrolidone/pyrrolidine

based temperature responsive polymer systems have been used, among others, and are extensively

reviewed elsewhere [23]. Other relevant biocompatible materials are the poly(ethylene glycol) (PEG)

(or poly(ethylene oxide) [23, 24]. The interest in such compounds arises from their versatility allowing

for the design of novel macromolecular architectures, and the possibility to access high-molecular-

weight PEG-based polymers using relatively mild synthesis conditions [25].

3

On the other hand, pH-responsive polymeric materials have received considerable attention for

enhanced diagnostics and therapeutic efficacy in the treatment of a wide range of diseases, due

to their ability to convert environmental pH stimulus to an observable change, such as solubility,

volume or chain conformation in aqueous solutions [26]. For instance, there has been much interest

in developing pH-responsive drug delivery systems to target or be triggered in the extracellular matrix

of solid tumor tissues [15]. Many of the reported pH-responsive polymers used as delivery systems

have pKa values lower than 6.0, including the acid-labile pH-responsive polymer systems [27, 28],

histidine-based systems [29], and 2-aminoethyl methacrylamide (AEMA)-based systems [30], which

are specially suitable for intracellular targeting and delivery after cellular uptake in non-cancerous

cells. However, the intracellular pH of many cancer cells is neutral or slightly alkaline relative to normal

cells, which have intracellular endosomes or lysosomes with a pH of 4.5-6.5 [31, 32]. Considering the

pH gradient difference between normal and cancerous tissue, pH-responsive polymer systems with a

pKa of 6.5-7.2 [31, 32] need to be developed to specifically target the weakly acidic extracellular matrix

microenvironment of solid tumors. Among the pH-responsive polymer systems with a pKa above 6.0,

tertiary amine AEMA-based polymers have been recognized as one of the most promising groups,

since their pKa can be tuned from approximately 4.5 to 8.5, depending on different substituent groups

[33].

When considering biomedical applications, it is often useful for more than one stimuli to be able

to cause polymer conformational changes, simultaneously. In fact, through block copolymerization,

where each type of monomer is arranged systematically in the form of blocks, it is possible to couple

a wide variety of different stimuli responsiveness on the same molecule, by the simple incorporation

of monomers with different properties. For instance, ionizable and temperature-responsive groups

may be coupled (Figure 1.2). Because biological systems possess different physiological microenvi-

roments, the possibility of joining virtually any combination stimuli-responsiveness represent an op-

portunity for the formulation of suitable DDS. Temperature and pH dual-responsive polymers have

been designed to improve drug release by the deformation and precipitation of core/shell nanoparti-

cles in acidic tumor microenvironments [34, 35]. For example, it was reported that temperature- and

pH-responsive block copolymer micelles, containing poly(ε-caprolactone) and aminoacid functional-

ized poly(triethylene glycol) loaded with doxorubicin (DOX), exhibited greater anti-tumor activity and

higher release efficacy in acidic environments than did free DOX in xenograft tumor models, mainly

due to the pH-responsive phase transition of this system, which enhanced drug release in the acidic

physiological environments typically present in tumors [36]. Other groups have constructed tempera-

ture and pH dual-responsive nanoparticles by copolymerizing N-isopropylacrylamide with acrylic acid,

methacrylic acid (MAA), and N,N-dimethylarylamide [37–39], indicating that the loading capacity and

release kinetics of drugs can be modulated by varying the structural and physicochemical properties

of the constituent block copolymers. Thus self-assembled structures based on dual-stimuli responsive

block copolymers are promising candidates for effective DDS.

4

Figure 1.2: Schematic illustration of a diblock copolymer self-assembly and dissociation in response to changesin pH or temperature. (Adapted from [40]).

1.2 Background

1.2.1 Amphiphilic block copolymers: self-assembly

Polymers permit the length scales to be greatly varied, the superstructure to be controlled, and

specific functions to be performed. The simplest primary structure which allows these functions to

be carried out are diblock copolymers, which are macromolecules consisting of one polymer chain

made of two blocks, each with a different repeating unit covalently bound to each other. This covalent

junction and the different chemical nature of the two blocks leads to the ability of self-assembling into

a variety of morphologies. The length scale of these ordered structures matches the one of the poly-

mer chains themselves, which generally range from 1-100 nm. These morphologies can be formed in

the bulk state or in solution. For the purposes of the present work, only the latter will be addressed.

The behavior of block copolymers in solution depends not only on the interaction between the two

blocks, but also on their interaction with the solvent, usually water in biomedical applications. If the

solvent is selective for one block, ordered structures will be formed with one block composing the

water soluble exterior, and the other being located in the interior, protected from the solvent. In this

scenario, where one block is hydrophobic and the other is hydrophilic, the copolymer is referred to as

being amphiphilic. However, this definition should not be oversimplified, as self-assembled structures

could arise, for example, from two water-soluble polymer blocks if they feature a sufficiently large

difference in hydrophilicity ”degree”. Spontaneous self-assembly is driven by the natural tendency

of the system to minimize its energy within a given solvent [41]. Thus, these systems have been

proven to be of interest as they can create structures such as micelles - a sheltered hydrophobic core

sterically stabilized by a hydrophilic corona, able to accommodate therapeutic poorly water-soluble

compounds. Block copolymers self-organize into diverse micellar structures above a certain concen-

tration, which is called the critical micelle concentration (CMC) (Figure 1.3), or critical aggregation

concentration (CAC), if no defined structure is formed. Starting from low polymer concentration in

aqueous solution, single chains are molecularly dissolved in solution and are called unimers. In this

regime, the surface tension of an aqueous solution is high because the air/water interface is not

fully populated by the polymer chains and therefore, there is no formation of micelle. As the polymer

concentration increases, the surface tension at the water/air interface decreases until it reaches satu-

ration, turning micelle formation energetically favorable when the CMC is crossed. Generally, the CMC

depends on a variety of factors, such as the nature of the hydrophilic and the hydrophobic block, the

5

presence or absence of charged groups, that ultimately will depend on the pH and the ionic strength

of the solution, temperature, etc. As a general rule, an increase of either the hydrophilic or hydropho-

bic content leads to lower CMC values. Temperature also plays an important effect in CMC, due to

its influence on the water solubility of the two blocks. This feature is denominated critical micelle tem-

perature (CMT) or Krafft point - defined as the minimum temperature at which micelles are formed

[41]. From a thermodynamical perspective, micellization is well described by the closed association

mechanism, which is defined by a dynamic equilibrium, above the CMC, between dissolved chains

(i.e. unimers) and chains forming the micelles, where a constant exchange occurs (Figure 1.3).

Figure 1.3: Formation of micelles in a selective solvent for one of the blocks (top) and dependence of the unimerand micelle concentration as function of the polymer concentration in solution (bottom). (Adapted from [42, 43]).

Various micellar morphologies are accessible depending on the characteristics of the block copoly-

mer (Figure 1.5 (a)). Generally, when the hydrophilic block is longer than the core block, the shape of

the resulting micelles is spherical. Conversely, increasing the length of the core segment beyond that

of the corona-forming chains may create other non-spherical structures in solution, including rods,

vesicles and lamellae, among others, intensively reviewed elsewhere [44, 45]. The morphology of the

resulting micelles is a result of the inherent molecular curvature arising from the relative sizes of the

soluble and insoluble parts and of the way this influences the packing of the copolymer chains within

the aggregates. The packing parameter p expresses the ratio of the molecular volume of the insoluble

chain to the volume actually occupied by the copolymer blocks in the assembly, usually dictating the

6

most likely self-assembled structure. The balance between hydrophobic and hydrophilic interactions

gives rise to an optimal surface area, a0, of the insoluble block at the interface between the soluble

and insoluble blocksṪhis area, together with the length, lc, and the volume, v, of the insoluble block

contributes to the packing parameter, defined as:

p =v

a0lc(1.1)

(a)

(b)

Figure 1.5: Influence of the dimensionless packing parameter of polymer chains in the resulting self-assemblystructures. (a) Schematics of the curvature of the molecule estimated through the dimensionless packing pa-rameter. (b) Various self-assembled structures formed by amphiphilic block copolymers with different packingparameters. (Adapted from [44, 46]).

Regarding the biomedical use of block copolymers, the CMC value is also an indicator of the

ability to form micelles and for their stability: the lower the CMC value, the easier micelles are formed,

and the more stable they are. Consequently, to develop improved drug delivery systems, amphiphilic

block copolymers that are able to form more stable micelles with low CMC values are appropriate

candidates [47, 48].

1.2.2 Temperature-responsive polymers

As previously mentioned, the most commonly used mechanism in temperature-responsive poly-

mers, for biomedical proposes, is the polymer volume phase transition in solution. When a certain

7

temperature is crossed, it causes a sudden change in the solvation state of the polymer. Depending

on the behavior upon heating, temperature-responsive polymers can be divided into two categories

(Figure 1.6) - if phase separation occurs upon heating, the polymer is said to have a lower critical so-

lution temperature lower critical solution temperature (LCST), whereas if it becomes soluble, i.e. just

one single phase, with increasing temperature, the polymer presents a upper critical solution temper-

ature upper critical solution temperature (UCST). Thus, this phase transition classification concerns

the critical temperature points below and above which the polymer and solvent are completely mis-

cible, respectively. Despite UCST and LCST behavior concepts apply for both organic and aqueous

systems, only the latter will be considered as these are of interest for biomedical applications. It is

noteworthy to stress that UCST and LCST terms should be used when associated with a phase dia-

gram, being attributed to, respectively, the maximum or the minimum of that phase diagram, whereas

the temperatures on the phase boundary line are called cloud-point temperature (Tcp).

Figure 1.6: Schematic phase diagrams associated with LCST (lower critical solution temperature) and UCST(upper critical solution temperature) behavior. The blue line represents the phase separation boundary, whichresults in a cloud point. (From [49])

The majority of water-soluble polymers that possess a LCST undergo phase separation upon

heating, accompanied with a local structural transition involving water molecules surrounding cer-

tain regions of the polymer chains in solution. The common characteristic of temperature-responsive

hydrophilic homopolymers is the presence of hydrophobic groups such as methyl, ethyl, and propyl

groups [50]. The interactions that take place in an aqueous polymer solution are: polymer-polymer,

polymer-water, and water-water. For instance, there is a change in the hydration state of the polymer

chains, which is a result of competing interactions, where intra- and intermolecular interactions of the

polymer are favored compared to a solubilization by water. Phase transition then occurs as result of

the polymer collapse to a globule-like conformation, leading to the formation of polymer-rich droplets

and clouding of the solution [51].

1.2.2.A Collapse mechanism

As seen previously, temperature-responsive polymers with a LCST in water experience a collapse

for temperatures higher than the Tcp, becoming less hydrophilic as they change conformation from an

expanded (soluble) to a globular (insoluble) state, i.e. a coil-to-globule transition. More specifically, the

8

LCST transition reflects local structural transitions involving water molecules surrounding the polymer

monomers. At low temperatures, the polymer is hydrophilic as the water molecules are able to interact

with its polar groups - and obliviously to themselves - via hydrogen bonds, which is the initial driving

force for the polymer dissolution, thus the polymer chain features an extended coil conformation. At

higher temperatures, water molecules are released into the bulk, allowing interactions between the

newly exposed hydrophobic monomers, only slightly water-soluble, while trying to minimize the en-

tropic loss of the system. However, the detailed reasons for the collapse are still unclear [52, 53].

It has been theoretically predicted [54, 55] that a temperature increase gives rise to local collapsed

structures within the polymer chain which, consequently, adopts an intermediate necklace-like con-

formation (Figure 1.7) where hydrophilic groups are adjacent to single pearls of hydrophobic groups.

The water molecules bind to the polymer chain in sequences, a process called cooperative hydration

[54]. As temperature approaches Tcp from below, the average length of the sequence is sharply re-

duced. The thermodynamics of mixing and demixing of polymers in solution are thoroughly discussed

elsewhere [53].

Figure 1.7: Schematic of the cooperative hydration of LCST type temperature-responsive polymers. Sequentialhydrogen bonds form along the polymer chain due to the cooperative interaction between adjacent bound watermolecules. ξ: sequence length over which water molecules bind to the chain. The collapsed parts are marked bythin circles. (From [56]).

In this context, it is noteworthy to mention that often, amphiphilic polymers which remain soluble

in aqueous media after the transition to the globular state mimic proteins (eg. human hemoglobin),

earning the designation protein-like polymers [57]. In both cases, hydrophobic units are enclosed in

the core of the globule, while more hydrophilic ones form a shell around.

1.2.3 pH-responsive polymers

Along with temperature, pH is an important stimulus when considering biomedical applications,

which can be used through pH-responsive polymers whose solubility, volume, configuration and

conformation are able to be reversibly manipulated by changes in external pH [26]. Essentially, pH-

responsive polymers are polyelectrolytes that possess weak acidic (anionic) or basic (cationic) groups

9

that either accept or release protons in response to variations in the pH of the aqueous media. These

alter the ionic interactions, hydrogen bonding, and hydrophobic interactions between the polymer and

the surrounding water molecules resulting in a reversible microphase separation or self-organization

phenomenon [58]. In this context, polymers bearing basic moieties such as amine groups, will accept

protons and be positively charged (through protonation) under acidic conditions, thus the chains tend

to expand due to the electrostatic repulsions. Whereas under alkaline conditions, the basic groups of

the polymer release its protons, becoming uncharged (through deprotonation), leading to the collapse

of the chain in solution (Figure 1.8). The opposite behavior is found in polymers containing acidic moi-

eties, such as carboxyl acid groups, since the ionization of the acidic groups (through deprotonation)

will occur under alkaline conditions.

Figure 1.8: Schematics of the effects of the pH in solution in micelles composed by an hydrophobic core and apolybasic pH-responsive shell. (Adapted from [59]).

The pH range in which a reversible phase transition occurs is tailorable, for example by selecting an

ionizable moiety with a pKa matching the desired pH range. Moreover, the proper selection between

acid or base should be considered for the envisaged application. For instance, the mildly acidic pH

in tumor tissues (pH ~6.5-7.2) and in inflammatory tissues [31, 32], as well as in the endosomal

intracellular compartments (pH ~4.5-6.5) [60, 61] could be potential environments to trigger drug

release from pH-responsive DDS upon arrival at the targeted disease site.

1.3 Polymers under study

Taking into account the proven functionalities that temperature- and pH-responsive block copoly-

mer systems possess for biomedical application, such as drug delivery systems, in the present work

dual-responsive polymer sensitive to both temperature and pH are studied. Regarding tempera-

ture stimulus, previous research have indicated the potential of an emerging class of temperature-

responsive polymers that may compete with the well-studied pNiPAAm - polymers with PEG (poly(ethylene

glycol)) short side chains [25, 57, 62–64]. Polymerization of these monomers provides direct access

to previously inaccessible poly(ethylene glycol) (PEG) architectures, increasing the possibilities far

beyond linear PEG (Figure 1.10).

10

(a)

(b)

Figure 1.10: (a) Chemical structures of various oligo(ethylene glycol) methacrylates. Hydrophobic and hy-drophilic molecular regions are indicated in red and blue, respectively. Monomer unit used in the present work isindicated. (b) Molecular structure of standard linear PEG and nonlinear PEG-analogs (rights) from poly(ethyleneglycol)-containing monomers (left). (Adapted from [65]).

This kind of monomers combines the biocompatibility of linear PEG, which has been widely applied

in DDS, with the temperature-responsive properties of pNiPAAm with LCST behavior in aqueous

media. For instance, PEG-methacrylates such as 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA)

have been used, as a copolymer along with other PEG-methacrylate monomers, for drug delivery

applications [23, 66, 67]. In aqueous solution, this monomer features a Tcp = 26-27°C with very weak

hysteresis [68].

On the other hand, various ionizable monomers could be the starting point for pH-responsive DDS,

including sulfonates, carboxylic acids, and amines. The amine-containing 2-(diisopropylamino)ethyl

methacrylate (DPA) monomer, is a biocompatible [69] pH-responsive material with a pKa around

6.2-6.4 [33, 70], close to the values reported in literature on the pH in various cancerous microen-

vironments [71, 72]. In aqueous solution, when the pH is lower than the polymer pKa, the tertiary

amine groups are protonated (i.e. positively charged) and the monomers become soluble. When the

solution pH is higher than the monomer pKa, the monomers become less hydrophilic (i.e. uncharged)

due to deprotonation. Thus, DPA has been used as a comonomer to develop pH-responsive drug

delivery systems [73–75]. Consequently, a diblock copolymer bearing these two monomers (DPA

and MEO2MA) should feature different conformations depending on the conditions in solution. For

11

instance, the temperature-responsive PMEO2MA block is expected to be, respectively, expanded (hy-

drophilic) or collapsed (hydrophobic) in solution for temperatures below or above than the Tcp. The

pH-responsive PDPA block is expected to be, respectively, expanded (hydrophilic) or collapsed (less

hydrophilic) in solution, at pH values below or above than the pKa value of the PDPA block (Figure

1.12 (b)). Thus, as exemplified in Figure 1.12 (c), the PDPA-b-PMEO2MA diblock copolymer has

the potential to generate self-assembled structures in solution, such as core-shell micelles, under

conditions that make the solvent selective for one of the two blocks, such as (i) low pH and high

temperature: hydrophilic (positively charged) pH-responsive PDPA shell, surrounding a hydropho-

bic core of collapsed temperature-responsive PMEO2MA blocks; and (ii) high pH and low tempera-

ture: hydrophobic (uncharged) pH-responsive PDPA blocks form the core, being surrounded by the

hydrophilic temperature-responsive PMEO2MA blocks. Moreover, (iii) low pH and low temperature

should yield molecularly dissolved unimers, with both blocks being hydrophilic, whereas (iv) high pH

and high temperature should make both blocks hydrophobic, hence causing formation of aggregates.

In the first two cases ((i) and (ii)), the micelles formed could, ultimately, transport hydrophobic thera-

peutic cargo which would be released in the latter conditions ((iii) and (iv)).

(a)

12

(b)

(c)

Figure 1.12: (a) Chemical structure of the PDPA-b-PMEO2MA diblock copolymer. (b) Schematics of the ex-pected collapse behavior of the PDPA-b-PMEO2MA diblock copolymer in aqueous solution. (c) Schematics of theself-assembled structures potentially formed as a result of the aggregation behavior of the PDPA-b-PMEO2MAdiblock copolymer in aqueous solution. (Adapted from [15]).

13

1.4 Rationale and objective

Stimuli-responsive polymeric systems constitute an attractive option for enhancing diagnostics

and therapeutic efficacy. This is achieved through their ability to convert environmental stimuli to

an observable change, such as solubility, volume or chain conformation in aqueous solution. While

research on temperature and pH-responsive block copolymers, based on PEG-methacrylates and

amine-based methacrylates, has been increasingly developed, still few studies attempt to characterize

the underlying mechanism of the stimuli-responsiveness of these kind of block copolymers. In face of

the importance of the behavior of dual-stimuli responsive block copolymers in the creation of novel

and effective drug delivery systems, this project aims the study of the aggregation behavior of the

temperature- and pH-responsive PDPA-b-PMEO2MA diblock copolymer in aqueous solution, below

and above of both the cloud-point temperature of the LCST type PMEO2MA block and the pKa of the

basic PDPA block.

The first part of our work envisages the synthesis of the PDPA-b-PMEO2MA diblock copolymer,

achieved through reversible addition-fragmentation chain transfer (RAFT) polymerization, which pro-

vides a powerful synthetic tool regarding the synthesis of block copolymers. By means of 1H NMR

spectroscopy analysis, the resulting reaction products of each synthesis step are characterized. The

second part aims at the investigation of the aggregation behavior of the PDPA-b-PMEO2MA diblock

copolymer under different temperature and pH conditions in aqueous media. Fluorescence correla-

tion spectroscopy FCS provides an insight on the aggregation behavior of our polymer for different pH

conditions in solution at room temperature, in particular of the pH-responsive PDPA block, taking ad-

vantage of the resolution power of this technique at low concentrations, where individually solubilized

macromolecules are of interest. By adding a water-insoluble fluorescent dye (Rhodamine 6G) that

attaches to the hydrophobic domains present in the polymer, FCS technique enables the detection

of the formed structures and the determination of the correspondent hydrodynamic radii. Such infor-

mation is also obtained with temperature-resolved FCS (T-FCS), whose measurements, performed at

temperatures near the Tcp of the PMEO2MA block, allow for the evaluation of the influence of the tem-

perature on the aggregation behavior of our polymer, under different pH conditions. This technique

is utilized along with macroscopic methods such as turbidimetry, which helps to corroborate the as-

sessment of the Tcp of the PMEO2MA block, for the same pH conditions. Complementary knowledge

about the aggregation behavior in solution for different temperatures and pH values, is gained with

small-angle X-ray scattering (SAXS) analysis, which allows to for the characterization of the structure

properties of our polymer (such as size and conformation), at the local scale. Thereafter, with this

experimental work we attempt to better understand the mechanism of the stimuli-responsiveness of a

biomedical relevant diblock copolymer, able to respond simultaneously to temperature and pH.

14

2Methods

Contents2.1 Controlled/living radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . 162.2 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . 212.3 Small-Angle X-Ray Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

15

2.1 Controlled/living radical polymerization

Polymers are constituted by repeating units (i.e. monomers), which can be assembled in a wide

range of possibilities. Following the developments from early radical polymerization studies [76], pre-

cise control of polymeric structural parameters prepared by radical polymerization techniques is now

possible, giving rise to a virtually unlimited number of novel polymeric materials. Among others, stud-

ies conducted by Rizzardo and coworkers have given tremendous contribution in this field [77–79]. To

understand how the more recent forms of controlled/living radical polymerization (CRP) work, we first

need to consider the mechanism of the conventional process. Briefly, conventional free radical poly-

merization is a chain reaction. The chains are initiated by radicals (activated by an initiator) adding

to monomer. Chain propagation then follows a sequential addition of monomer units to the radical

so formed. Chain termination then occurs when propagating radicals react either by combination or

disproportionation (Figure 2.1).

Figure 2.1: Scheme for conventional radical polymerization (Pn - radical).

Despite its wide spread use in industry, short life-time chain growth and fast irreversible termina-

tion controlled by diffusion effects were translated in limitations, with respect to the control level of

the polymer structure and molecular weight distribution. These bottlenecks are avoided by using a

controlled/living radical polymerization. In an ideal living polymerization, all chains (i) are initiated at

the beginning of the reaction, (ii) grow at a similar rate, and (iii) survive the polymerization: there

is no irreversible chain transfer or termination. If initiation is fast compared to the propagation (i.e.

growth) of the chains, the molecular weight distribution is very narrow and chains can be extended

by further adding monomers into the reaction. Thus, living radical polymerization only becomes pos-

sible if the species (X) reacts with the propagating radicals (P∗n) by reversible deactivation (Figure

2.3 (a)) (only one propagating species) or reversible chain transfer (two different propagating species)

(Figure 2.3 (b)), so that the majority of chains are maintained in a dormant form (Pn-X) and do not

undergo radical-radical termination. A fast equilibrium change, between active and dormant forms,

allows for an intermittently chain growth. Under these conditions, molecular weight increases linearly

with conversion and the weight distribution is very sharp, which is not the case in the conventional

processes.

16

(a)

(b)

Figure 2.3: (a) Schematics for reversible deactivation and (b) reversible chain transfer.

Thus, and according to Arslan [80], CRP techniques are expected to display (i) first-order kinetic

behavior, (ii) pre-determinable degree of polymerization (DP), (iii) narrow molecular weight distribu-

tion and (iv) long-lived polymer chains. With this, the synthesis of complex polymers with diverse

topologies, and functional groups is achievable.

2.1.1 RAFT principle

Controlled/living radical polymerization (CRP) techniques such as nitroxide-mediated polymeriza-

tion (NMP), atom-transfer radical polymerization (ATRP), and reversible addition-fragmentation chain-

transfer (RAFT), had been worth of attention in current days [81]. Although these methods have

different initiation systems, they all take advantage of mechanisms depending on activation and de-

activation equilibrium [82]. Among the existing CRP techniques, RAFT polymerization is arguably the

most versatile process, conceding the benefit of being able to synthesize well-defined polymers for

a wider range of monomers under mild reaction conditions. Moreover, it is tolerant to a variety of

reaction conditions and functionalities, and can be performed using existing conventional free-radical

polymerization set-ups, such as solution, emulsion, and suspension polymerization [78, 79, 81, 83].

The key feature of the RAFT polymerization technique is the sequence of addition-fragmentation

equilibrium (Figure 2.4), that relies on the use of compounds employed as chain-transfer RAFT

agents, or, more precisely, chain transfer agent (CTA) - organic compounds containing a thiocar-

bonylthiol (ZC(=S)SR)) moieties. Commonly used RAFT agents include thiocarbonylthio compounds

such as dithioesters, dithiocarbamates, and trithiocarbonates. Its purpose is to reversibly deactivate

propagating radicals in a way that the majority of living chains are maintained in a dormant state and

to create a rapid equilibrium between the active and dormant chains - the R group initiates the growth

of polymeric chains, while the Z group activates the thiocarbonyl bond towards radical addition and

stabilizes the intermediate radical. The structures of the R and Z groups are of critical importance to a

successful RAFT polymerization. Also, the R group of a RAFT agent is important in the pre-equilibrium

stage of the polymerization. The R group should feature faster kinetics than the propagating radical

and must efficiently reinitiate monomer as an expelled radical [79, 84].

17

Figure 2.4: Mechanism of RAFT polymerization and overall reaction. The first step of polymerization is theinitiation step, where a radical is created (step i). The oligomeric radicals produced in the initiation step react withthe RAFT agent (1) in the initial step (ii). The radical intermediate (2) can fragment back to the original RAFTagent (1) and an oligomeric radical or fragment to yield an oligomeric RAFT agent (3) and a reinitiating R radical.The structure of R should be such that it is a good reinitiating group. It should also fragment at least as quicklyas the initiator or polymer chains from the stabilized radical intermediate (2). Following re-initiation, polymerchains grow by adding monomer (step iii), and they rapidly exchange between existing growing radicals (as inthe propagation step) and the species capped with a thiocarbonylthio group (step iv). The rapid interchangein the chain-transfer step via the formationof intermediate 4 limits the termination reactions. Although limited,termination reactions still occur via combination or disproportionation mechanisms (step v). (Adapted from [85]).

18

In practice, RAFT polymerization finds its use as a tool for synthesizing block copolymers and com-

plex polymers with well-defined structures. Block copolymers may be formed by the chain extension

of a homopolymer with a second monomer (Figure 2.5). Generally speaking, the product obtained by

homopolymerization of the first monomer is typically isolated, and the chain extension of the resulting

polymer is conducted afterwards with a second monomer in the presence of an appropriate initiator.

In block copolymer synthesis by RAFT polymerization, the order of the blocks is of importance. The

macro-CTA, S=C(Z)S - Pn, in which the Pn block corresponds to the initial homopolymer (poly(M1))

and Z is the stabilizing group, should have a high transfer rate in the subsequent polymerization of

the second monomer (M2) to give the poly(M2) block. This requires that the leaving ability of the

propagating polymer radical (Pn) must be greater than, or at least comparable to, that of the second

polymer radical (M2) under the reaction conditions. A fast conversion of the macro-CTA to a block

copolymer is also required to achieve a low polydispersity, as it allows all the M2 blocks to be initiated

at approximately the same time [85, 86]. When the appropriate parameters are achieved, RAFT poly-

merization can be used for a broad range of applications, namely the synthesis of block copolymers

able to self-assemble into organized structures such as micelles, suitable for drug delivery purposes

[84, 87, 88].

Figure 2.5: Mechanism of block copolymer synthesis through the RAFT technique. (i) Overall reaction and (ii)mechanism of RAFT polymerization for the synthesis of diblock copolymers - M1 and M2 are different monomers.(Adapted from [85]).

2.1.2 Experimental

Reagents: 2-(N,N-diisopropylamino)ethanol (Sigma-Aldrich, 98%), methacryloyl chloride (Fisher

Scientific, 97%), triethylamine (TEA) , tetrahydrofuran (THF) (Scharlau, 99%), hydroquinone (Sigma-

aldrich, 99%), 1,4-dioxane (Scharlau, 99.5%, stabilized with 2.5 ppm of 2,6-di-tert-butyl-4-methylphenol),

dichloromethane (Sigma-Aldrich, 96%), 2,2-azobis(2-methyl-propionitrile) (AIBN) (Sigma-Aldrich, 99%),

dimethylformamide (DMF)(Scharlau, 99.8%), trifluoroacetic acid (TFA) (Sigma-Aldrich, 99%), ethanol

(Sigma-aldrich, 99.8%) and sodium sulfate (Panreac, 99%). The CTA/RAFT agent 3-(benzylsulfanylthiocarbonylsulfanyl)-

propionic acid ) was synthesized according to the literature procedures [89].

DPA monomer was synthesized by a one-step reaction between 2-(N,N-diisopropylamino)ethanol

19

and methacryloyl chloride. The synthesis was achieved as follows: hydroquinone (0.20 g) and an-

hydrous THF (100 mL) were added into a two necked flask, closed with rubber septa to minimize

oxygen and humidity intake. After the flask was put under argon atmosphere, a solution of 2-(N,N-

diisopropylamino)ethanol (2.4 mL, 14 mmol, 1 eq) and anhydrous TEA (2 mL, 0.014 mmol, 1 eq) were

added. After cooling down the reaction to 0°C, methacryloyl chloride (1.4 mL, 14 mmol, 1 eq) was ad-

ministrated dropwise. The resulting solution was refluxed, under stirring, in an oil bath (80°C) for 2h,

and filtered afterwards to remove the precipitated TEA-hydrochloric salt, which was rinsed with THF.

The THF was removed from the filtrate using a rotary evaporator and the resulting residue was diluted

with dichloromethane, washed with water, 10wt% potassium carbonate solution, saturated NaCl so-

lution, and finally with water. The organic layer was collected and dried under agitation with sodium

sulfate. After filtration of the salt, dichloromethane was removed using a rotary evaporator and the

residue was distilled under reduced pressure to give pure DPA as an transparent oil (1.8g, 8.3 mmol).

For the preparation of DPA homopolymer (PDPA), which served as a macro-CTA for the subse-

quent block copolymerization, CTA (24 mg, 0.087 mmol), AIBN (1.45 mg, 0.009 mmol), DPA (0.82 g,

3.77 mmol, 1eq), TFA (0.48 mg, 3.77 mmol) and ethanol (3 mL) were introduced into a Schlenk tube.

The flask was sealed with a rubber septum, subsequently degassed through freeze pump cycles with

liquid nitrogen, and left under argon atmosphere. Afterwards, the tube was heated (70°C) under stir-

ring for 24h. The polymerization was terminated by quenching in liquid nitrogen. The resulting solution

was concentrated. The product was precipitated into methanol, and dried under vacuum.

Block copolymerization was prepared by adding 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA,

Sigma-Aldrich, 95%) monomer to the PDPA homopolymer that served as macro-CTA . The synthesis

of the diblock copolymer PDPA-b-PMEO2MA was proceeded within the work group (Lisbon, Portugal),

similarly to the synthesis of the PDPA homopolymer, according to the literature procedures [70].

The resulting products of DPA monomer synthesis, PDPA homopolymer, and PDPA-b-PMEO2MA

diblock copolymer polymerization were analyzed by 1H nuclear magnetic resonance (1H NMR) spec-

troscopy (Bruker Avance II 400 MHz, UltraShield Magnet - Lisbon, Portugal) in CDCl3 at room tem-

perature, to determine the reaction conversions, the degree of polymerization (DP) and the number

average molecular weight (Mn) through processing of the resulting 1H NMR spectra with MestReNova

software [90]. The requisite peak areas were obtained by numerical integration of the peaks present

in the 1H NMR spectra, after the assignment of each spectrum and identification of the correspondent

peaks. In order to obtain a reliable baseline for the numerical integration, the signal of the isolated

resonance of deutered chloroform (CDCl3 - 7.260 ppm) was chosen as a reference peak.

2.1.3 Data analysis

In order to confirm that the product of each step of the polymerization reaction performed is the

envisaged one, 1H NMR analysis was performed. Regarding homopolymerization, whether of the

PDPA or the PMEO2MA block, when the monomer is consumed during the polymerization, the vinyl

protons from the methacrylate moiety, are used for the covalent bonds between adjacent monomers,

and therefore the vinyl peaks become weaker, whereas protons from the functional side group remain

20

as the conversion to polymer proceeds. From the 1H NMR spectra obtained, the degree of conversion

was estimated by comparing integrated peak intensity of the vinyl protons of unreacted monomers, to

the integrated peak intensity of protons from the side groups, present in both the monomer and the

polymer [91].

conversion(%) =(

1− number of protons of resonancetotal integration area of monomer + polymer

)× 100 (2.1)

Another important characterization parameter is the DP, which gives the number of the repeat-

ing units from each polymerization reaction. Here, the DP was estimated assuming (i) a CTA/initiator

molar ratio high enough (in our case, [CTA]/[AIBN] = 10) to consider that every CTA molecule is in-

corporated into each initiated polymer chain, and that (ii) every growing chain undergoes an efficient

polymerization via the RAFT mechanism. As a result, the DP depends only the monomer and CTA ini-

tial concentrations ([Monomer]0 and [CTA]0, respectively), and on the fraction of converted monomer,

as follows:

DP =[Monomer]0

[CTA]0× conversion(%) (2.2)

Consequently, the DP was used to estimate the number average molecular weight (Mn) (Equation

2.3), by taking into account the molecular weight of the polymerized DPA monomer (M(Monomer))

and the molecular weight of CTA (M(CTA)).

Mn = DP ×M(Monomer) +M(CTA) (2.3)

Moreover, because the diblock copolymer was synthesized using the PDPA homopolymer as

macro-CTA, its molecular weight (M(PDPA)) was used instead of M (CTA), to give the total number

average molecular weight of the PDPA-b-PMEO2MA diblock copolymer, as follows:

Mn,total = DP ×M(Monomer) +M(macro-CTA) (2.4)

2.2 Fluorescence Correlation Spectroscopy

The underlying principle of fluorescence correlation spectroscopy (FCS) is based on the moni-

toring of fluctuations of the fluorescence intensity originating from species diffusing through a very

small detection volume. The fluctuations can be quantified in their strength and duration by tempo-

rally autocorrelating the recorded intensity signal, a mathematical procedure that gave the technique

its name. Essential information about the processes governing molecular dynamics of the diffusion

species can be derived from the temporal pattern by which fluorescence fluctuations arise and decay.

In general, all physical events that give rise to fluctuations in the fluorescence signal are accessible by

FCS. Consequently, this technique is useful to study a broad range of systems through the detection

of intensity variations arising from diffusional, rotational, and photochemical/physical processes of the

21

fluorescently labeled particles within the detection volume. This yields information, for example, about

the local concentrations, diffusion coefficients or characteristic of inter- or intramolecular interactions

of those particles. The idea of fluorescence correlation spectroscopy was originally introduced in the

early seventies by Weeb, Magde and Elson [92, 93] who used it to estimate the kinetics of DNA-dye

interactions. Around the same time, Rigler et al. suggested the use of fluorescence intensity fluctua-

tions to gain knowledge about rotational molecular motions and fluorescence life times [94]. Despite

showing great promise since its early stages, the FCS technique did not became widespread readily,

mainly due to issues concerning large detection volumes (leading to too high background noise), for

which longer measurement times and higher dye concentrations were required to compensate for the

low signal detected. Only later, with the introduction of extremely small detection volumes defined by

diffraction-limited laser beams along with confocal observation, by Rigler et al. [95, 96], a substan-

tially higher signal-to-noise ratio through better suppression of background radiation was achieved.

Further improvements concerning the quality of confocal optics, the time-resolution and sensitivity

of the detectors as well as the use of band-pass filters for better signal/noise discrimination lead to

single-molecule detection sensitivity [97].

2.2.1 FCS setup

As mentioned above, FCS is a temporal correlation analysis of the intensity variations which arise,

for instance, from the fluctuations of the concentration of fluorescent particles, entering or leaving

the detection volume, or from changes in their fluorescence properties. In this context, to extract

full potential from FCS technique, the concentrations and detection volume should be as small as

possible, such that only few molecules are simultaneously detected and, at the same time, increase

the fluorescence photon yield per single molecule [97]. A typical setup for fluorescence correlation

spectroscopy closely resembles that of a classical confocal microscope (Figure 2.6). A high numerical

aperture - ideally NA >0.9 - microscope objective (typically water or oil immersion) is used to focus

a laser beam to a diffraction limited spot into the sample. The resulting fluorescence light is then

collected by the same objective. After passing through a dichroic mirror and an emission filter, it is

imaged onto a confocal pinhole, which improves vertical axis resolution by blocking the fluorescent

light not originating from the focal plane. In this way a very small detection volume of less than 1 µm3

is created. The detection is done by a fast and sensitive detector, typically a single photon counting

avalanche photo diode (APD), that possess good quantum yield, and ultimately can reach the photon

detection resolution down to picosecond range.

22

Figure 2.6: (Left) Experimental setup for FCS. (a) A laser beam is first expanded by a telescope (L1 and L2),and then focused by a high-NA objective lens (OBJ) into a fluorescent sample (S). The fluorescence is collectedby the same objective, reflected by a dichroic mirror (DM), focused by a tube lens (TL), filtered (F), and passedthrough a confocal aperture (P) onto the detector (DET). (b) Magnified focal volume (green) within which thesample particles (black circles) are illuminated. (Right) (c) A typical fluorescence signal, as a function of time,measured for rhodamine green (RG) with a wavelength of 488 nm. (d) Portion of the same signal in panel c,binned, with an expanded time axis and average fluorescence F̄ . The signal is correlated with itself at a latertime (t + τ) to produce the autocorrelation G(τ). (e) Measured G(τ) describing the fluorescence fluctuation ofRG molecules due to diffusion only as observed by FCS. (Adapted from [98]).

These experimental arrangements, combined with the high sensitivity of the modern detection

systems result in several unique properties of FCS, namely, (i) the possibility of small concentrations

of the fluorescent species to be well below 1 part/fL, (ii) the access to diffusion coefficients in the

range 10-9 to 10-15 m2/s, (iii) the size of the fluorescent species can be as small as 1 nm, and (iv)

the small confocal volume can be positioned on in a specific place in a heterogeneous system, e.g.

inside a porous network. Despite of its high sensitivity and versatility, the FCS technique has also

some intrinsic technical problems and limitations. For example, precise measurements of the diffu-

sion coefficient and the concentration of the fluorescent species are possible only if the shape and

the exact dimensions of the confocal detection volume are well known [99]. Typically its dimensions

are determined by calibration measurements with a reference fluorescent dye with known diffusion

coefficient, e.g. Rhodamine 6G (Rh6G) [100].

23

Figure 2.7: Scheme of the chemical structure of Rhodamine 6G.

2.2.1.A FCS for micellization and aggregation studies

FCS is often used for the characterization of the micellization and aggregation of amphiphilic

block copolymers in solution, taking advantage of the present hydrophobic domains, on which the

highly hydrophobic dye Rh6G molecules associate. Consequently, this makes FCS suitable for the

detection of critical micelle concentration [101–103] of such polymers. This is possible because of

the difference in the diffusion time featured by Rh6G molecules below and above the CMC. In the

former, R6hG molecules interact with molecularly dissolved chains, which diffuse rapidly, in contrast

to the latter case, when they associate with the hydrophobic domains of larger self-assembled parti-

cles which moves slower, thus featuring lower diffusion coefficient. Moreover, through the recorded

diffusion time, the diffusion coefficient can be known, and therefore the hydrodynamic radius of the

micelles/aggregates can be determined, using the Stokes-Einstein equation, as discussed in the next

sections.

2.2.2 Experimental

FCS measurements were performed using a ConfoCor2 from Carl Zeiss Jena GmbH. An Ar+

laser operated at 488 nm, a motorized pinhole of diameter 80 µm, a C-Apochromat 40x/1.2 water

immersion objective, a BP 530-600 emission filter and an HFT 488 plate beam splitter. A Lab-tek

8-well chambered coverglass from Nalge Nunc International was used as a sample chamber. Each

measurement (run) time was 60 seconds, and the measurements were repeated 10-15 times, and the

correlation functions were averaged. The laser power was attenuated to 0.1-1.0% of maximum power

(200mW) to avoid bleaching of the dye probe. The count rate was maintained above 5 kcounts/s,

usually 5-15 kcounts/s. The resulting autocorrelation curves were fitted using Equation 2.5 [104], and

were normalized with respect to the lowest value of the fitted range (τ = 0.004 ms).

G(τ) = 1 +1

N

[1 +

TT1− TT

exp

(τ

τT

)] n∑i=1

ρi(1 +

τ

τD,i

)(1 +

1

(z0/w0)2τ

τD,i

) 12

(2.5)

In Equation 2.5, N is the total number of fluorescent particles in the detection volume, n the

number of different fluorescent species, τD,i the diffusion time of the i th species, ρi the amplitude

24

of the i th species, and z0 and w0 the half-height and half-width of the detection volume, respectively.

TT and τT are the triplet fraction and time, respectively. From the fit of 1 and 2 difussing species (n

= 1 and 2 in Equation 2.5), the triplet parameters were found to be TT = 0.1-0.2 and τT =1-3 µs. w0

was determined by measuring the diffusion time of Rhodamine 6G - τD,Rh6G - (Sigma-Aldrich, DRh6G

= 2.8×10-10 m2·s-1 [92]), and by using the relation:

w0 =√

4DRh6G · τD,Rh6G (2.6)

A value w0 ∼= 0.2 µm was obtained. The aspect ratio of the confocal volume, z0/w0 (otherwise

known as structure factor S, which describes the axial ratio of the detection) was determined from the

fit of the Rh6G correlation function in water and was typically 6.

To study the influence of the pH at room temperature, on the aggregation behavior of the polymer

under study by means of FCS, a stock solution of the PDPA-b-PMEO2MA diblock copolymer (4.5

mg/mL) was prepared in glass vials by dissolving the polymer in deionized water, for subsequent

dilution upon preparation of 1.5 mL samples of concentration ranging from 0.01 to 1.2 mg/mL. An

aliquot of the fluorescent dye Rh6G was added to the prepared polymer sample solutions and to the

control sample (deionized water with Rh6G), in such way that the final concentration of Rh6G was

4.79×10-7 mg/mL (10-9 M) to avoid signal saturation upon FCS analysis. After being briefly agitated,

the pH of the solutions was adjusted dropwise, by adding a strong acidic/alkaline aqueous solution,

so the sample concentration would not change significantly (10 µL of 0.1 M solution of either HCl

or NaOH). The deionized water, HCl and NaOH solutions were filtered (Rotilabo syringe filter; pore

diameter, 0.22µm) to remove dust particles, before the addition of the polymer and the Rh6G dye. The

pH was measured with a pH meter carefully cleaned with precision tissues to avoid contamination of

the sample by dust or other impurities. Using this sample preparation routine, 4 series of polymer

solutions were prepared having concentrations between 0.01 to 1.2 mg/mL and pH 3, 6, 7 and 10,

taking into account that the pKa of the pH-responsive block monomer DPA is ~6.4. The solutions were

agitated overnight and their pH measured afterwards. Any substantial deviation from the envisaged

pH value was corrected by adding more HCl or NaOH solution and the samples were agitated again

overnight.

To estimate the interesting temperature range, the polymer cloud-point temperature at different pH

values was assessed through turbidimetry measurements, at a wavelength of 500 nm (Varian Cary

50 UV-Vis photo spectrometer equipped with a temperature controller Varian Cary, Single Cell Peltier

Acessory). The polymer was dissolved in dionized water at pH 3, 6, 7 and 10, adjusted dropwise

using 0.03M HCl/NaOH solutions, resulting in four samples with a polymer concentration of 2 mg/mL.

For each sample the temperature was increased in steps of 0.1°C at a rate of 0.1 °C.min-1 followed

by a 10 min equilibration time. The resulting curves were normalized with respect to the transmittance

values recorded for the initial temperature (20°C), for each pH condition. The cloud-point temperature

was defined to be the highest temperature value matching a transmittance of 100%.

In order to evaluate the influence of temperature and pH on the aggregation behavior of the di-

25

block copolymer, temperature-resolved FCS experiments were performed at different pH values. The

same technical parameters used in FCS measurements at room temperature (such as run time, laser

power, etc). However, instead of the usual 8-well sample holder chamber used at room temperature,

a custom made sample holder (Figure 2.8) able to accommodate a electrical conducting indium-tin-

oxide (ITO) coated glass cover slips (8-12Ω, Spi Supplies) was used. The sample temperature was

installed and controlled using a voltage generator coupled with a temperature control system (ther-

mostat) [105]. Briefly, a voltage was imposed onto the ITO cover slip, forcing a current flow through

it, causing an increase of its temperature. The temperature was measured by a Pt100 platinum resis-

tance thermometer and controlled in the same fashion as a thermostat.

Figure 2.8: Sketch of the temperature-resolved FCS frame setup. (a) Top view, (b) Side view. A potential dif-ference is imposed on the copper pads leading to current passage through the ITO glass which in turn heatsup. The temperature is controlled by negative feedback around a given value defined by the user. The sameapplies for the heated oil-immersed objective. A PVC box serves as a protection from both the laser and thermalinsulation when filled with foam. (Dimensions not to scale).

The temperature is set by the user and fluctuates by ±0.2°C around the mean value. Several

other steps were performed to optimize the process: (i) placing of thermopaste onto the contact point

between the Pt100 sensor a

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