Photoinduced transfer processes in complex carrier systems for photodynamic therapy [Elektronische Ressource] / von Martin Regehly

Photoinduced transfer processes in
complex carrier systems for
photodynamic therapy
DISSERTATION

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Physik
eingereicht an der
Mathematisch-Naturwissenschaftliche Fakultät I
Humboldt-Universität zu Berlin
von
Dipl.-Phys. Martin Regehly
geboren am 02.04.1978 in Berlin
Präsident der Humboldt-Universität:
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Christian Limberg
Gutachter: 1. Professorin Beate Röder (HU-Berlin, Deutschland)
2. Professor Jürgen Raabe (HU-Berlin, Deutschland)
3. Professor Hiroshi Maeda (Sojo-Universität, Japan)

Datum der Promotion: 16. 07. 2008

Dedication
Dedication

For my family and my fiancée, who offered me love and support throughout the course of this
thesis.
2 Contents
Contents
1 INTRODUCTION 8
2 PHOTODYNAMIC THERAPY 10
2.1 PRINCIPLE OF PHOTODYNAMIC THERAPY 10
2.2 PHOTOPHYSICAL PROCESSES OF PHOTOSENSITIZATION 11
2.3 SINGLET MOLECULAR OXYGEN 14
2.4 FACTORS AFFECTING PDT EFFICIENCY 15
2.5 MACROMOLECULAR CARRIER SYSTEMS 17
2.5.1 Active targeting 17
2.5.2 Passive targeting 18
3 AIM OF THE WORK 21
4 EXPERIMENTALS 23
4.1 METHODS 23
4.1.1 Steady state absorption and steady state fluorescence spectroscopy 23
4.1.2 Fluorescence lifetime measurements by TCSPC 23
4.1.3 Steady-state singlet oxygen luminescence spectroscopy 25
4.1.4 Time-resolved singlet oxygen luminescence 27
4.1.5 Laser flash photolysis 28
4.1.6 Picosecond transient absorption spectroscopy (ps-TAS) 31
4.2 MATERIALS 33
4.2.1 P6, FHP6 and FP6 33
4.2.2 PEG-Zinc protoporphyrin 33
4.2.3 SMA-Zinc pr34
5 THEORETICAL FUNDAMENTALS OF PHOTOINDUCED TRANSFER
PROCESES 35
5.1 ENERGY TRANSFER PROCESSES 35
5.1.1 Exciton theory for molecular dimers 36
5.1.2 Förster Resonance Energy Transfer (FRET) 38
5.2 ELECTRON TRANSFER PROCESSES 41
5.2.1 Rehm-Weller Equation 41
5.2.2 Marcus Theory 43
3 Contents
6 HEXAPYROPHEOPHORBIDE A-FULLERENE [C ] MOLECULES AS PART 60
OF MODULAR CARRIER SYSTEMS 46
6.1 INTRODUCTION 46
6.2 PHOTOPHYSICAL PARAMETERS OF FHP6, P6, FP6 48
6.2.1 Steady state absorption spectra 48
6.2.2 Steady-state fluorescence 49
6.2.3 Time-resolved fluorescence 50
6.2.4 Steady-state singlet oxygen generation 51
6.2.5 Transient Absorption Spectroscopy 52
6.2.6 Compilation of photophysical parameters 55
6.3 MOLECULAR MODELING 56
6.4 PHOTOINDUCED ENERGY TRANSFER PROCESSES IN P6, FHP6, FP6 57
6.4.1 Förster resonance energy transfer in P6, FHP6 and FP6 57
6.4.2 Excitonic interactions in P6, FHP6 and FP6 59
6.5 PHOTOINDUCED ELECTRON TRANSFER PROCESSES IN FP6 59
6.6 NON-RADIATIVE RELAXATION PROCESSES IN P6, FHP6 AND FP6 62
6.7 MACROSCOPIC MODEL FOR THE TRANSPORT PROCESSES IN P6, FHP6, AND FP6 63
6.7.1 The model 63
6.7.2 Formal treatment and solution of the differential equation system 65
6.7.3 Analysi 69
6.8 CELLULAR UPTAKE AND PHOTOTOXICITY 71
6.9 CONCLUSIONS 72
7 POLY (ETHYLENE-GLYCOL) BASED POLYMER CARRIERS: PEG-ZNPP
75
7.1 INTRODUCTION 75
7.2 PHOTOPHYSICAL CHARACTERIZATION 77
7.2.1 Steady-state absorption spectra 77
7.2.2 Steady-state fluorescence 78
7.2.3 Time-resolved fluorescence 79
7.2.4 Time-resolved singlet oxygen emission 80
7.3 LASER FLASH PHOTOLYSIS 81
7.3.1 Measurements in solution 82
7.3.2 In vitro studies 83
4 Contents
7.4 PDT RELEVANT BIOLOGICAL ACTIVITY OF PEG-ZNPP 84
7.5 CONCLUSIONS 85
8 BLOCK-COPOLYMER MICELLAR CARRIERS: SMA-ZNPP 89
8.1 INTRODUCTION 89
8.2 PHOTOPHYSICAL CHARACTERIZATION 90
8.2.1 Steady-state absorption spectra 90
8.2.2 Steady-state fluorescence 92
8.2.3 Time-resolved fluorescence 93
8.2.4 Time-resolved singlet oxygen emission 94
8.3 LASER-FLASH PHOTOLYSIS 95
8.3.1 Measurements in solution 95
8.3.2 In vitro studies 96
8.4 CELLULAR UPTAKE AND PHOTOTOXICITY ON JURKAT LEUKEMIA CELLS 98
8.5 CONCLUSIONS 99
9 SUMMARY / ZUSAMMENFASSUNG 102
10 REFRENCES 109
11 PUBLICATIONS 130
12 ACKNOWLEDGEMENTS 131
13 CURRICULUM VITAE 132

5 Abbreviations
Abbreviations
1O Singlet oxygen 2
3O Ground state molecular oxygen 2
CMC Critical micelle concentration
DMF Dimethylformamide
DPBF 1, 3-diphenylisobenzofuran
EPR Enhanced penetration and retention
ET Electron transfer
EtOH Ethanol
FRET Förster resonance energy transfer
Ge Germanium
Hp Hematoporphyrin
HPMA N-hydroxy-propyl methacrylate
ISC Intersystem crossing
MCS Modular carrier system
MW Molecular weight
NCS Neocarzinostatin
NIR Near infrared
-O Superoxide ion 2
OD Optical density
PDT Photodynamic therapy
PEG Polyethylene glycol
PhCN Benzonitrile
PIC Photosensitizer immunoconjugate
PS Photosensitizer
RB Rose Bengal
ROS Reactive oxygen species
S First excited singlet state 1
SCE Saturated calomel electrode
SMA Styrene maleic acid
T First excited triplet state 1
TAS Transient absorption spectroscopy
TCSPC Time-correlated single photon counting
6 Abbreviations
TP Two-photon
TPA Two-photon absorption
ZnOEP Zinc octaethylporphrin
ZnP Zinc porphyrin
ZnPP Zinc protoporphyrin
Fluorescence quantum yield Φ F
Intersystem crossing quantum yield Φ ISC
Singlet oxygen quantum yield Φ Δ
Fluorescence lifetime τ F
Triplet state lifetime τ T
Singlet oxygen lifetime τ Δ


7 1. Introduction
1 Introduction
Cancer is one of the leading causes of death in western countries, and its incidence has been
rising. [Fer07, Jem07] Established cancer treatments, such as cytostatic chemotherapy,
radiation, and surgery are often inadequate for full recovery and they also have serious side
effects.
Photodynamic therapy (PDT) is an alternative non-invasive modality to treat cancers of the
head and neck, brain, lung, pancreas, intraperitoneal cavity, breast, prostate and skin. Today
PDT is not only approved for tumor therapy, it is used to treat several other diseases like age-
related macular degeneration, psoriasis and scleroderma as well. [Dol03, Hol03, Ber05]

PDT combines the use of photosensitizing dye molecules (photosensitizer), light and oxygen
to selectively destroy abnormal tissue. The photosensitizer, after activation with light of
appropriate wavelength, generates reactive oxygen species, mainly singlet oxygen. This
cyctotoxic effector destroys the tissue only in the local environment of the photosensitizer
leading to a defined destruction of the area where light has been applied. [Röd00a, Röd04]

In order to achieve effective treatments with low associated side effects, the photosensitizer
should selectively accumulate in the tumor tissue following administration to the patient. Up
to now, photosensitizers approved for clinical use display only a limited tumor specifity.
[Vro99, Don04] For this reason molecular delivery systems utilizing different targeting
strategies have been taken into consideration. [Mae02, Yok05, Dun06] Among them are
liposomes [Jia98, Der04, Sch05], protein nanoparticles [Pol02, Taw06], dendrimers [Bat01,
Nis03, Pau03], polymeric micelles [Zha03a, Rob06, Nos04] and photosensitizer-
immunoconjugates employing antibodies as targeting units.[Car01, Sav03, Ran07b]

Most of these targeting systems cause selective accumulation in tumor cells. In contrast, the
photodynamic activity of the photosensitizer-carrier complexes was significantly affected
compared to the photosensitizer alone. [Ham03, Sav03, Ran05b] It has been found that after
photoexcitation complex energy transfer mechanisms and nonradiative deactivation processes
take place among photosensitizers attached to or incorporated in such carrier systems.
[Erm05, Hac05, Hel05] These processes influence the potential of photosensitizer-carrier
conjugates to generate cyctotoxic oxygen species. As a consequence, the macromolecules
may be inefficient for the use in PDT.
8 1. Introduction
The present work investigates three promising photosensitizer-carrier systems regarding to the
photophysical processes after light excitation and their implications for the applicability of the
complexes for PDT. It is expected that the carrier system itself and the molecular architecture
of the conjugates have an important influence on the type and efficiency of intramolecular
deactivation or transfer processes. Revealing the underlying structure-property relationships
may improve the development of novel PDT agents.

Among the macromolecules studied, the focus is set on the examination of
hexapyropheophorbide a-fulleren