Determining the state of radionuclides in polymersomes using perturbed angular correlation

79 Pages·2015·2.84 MB·English

by  Laura Bogers

Determining the state of radionuclides in
polymersomes using perturbed angular
correlation spectroscopy

Stephanie Laura Christina Bogers

Master thesis performed at:

Section Radiation and Isotopes for Health
Radiation, Radionuclides & Reactors
Faculty of Applied Sciences
Delft University of Technology

In partial fulfilment of the requirements for the Degree of Master of Science
in Biomedical Engineering, track Medical Physics of the Delft University of Technology.
July 2015

Under the supervision of: Ir. R.M. de Kruijff
F.D.P. Geurink
Ing. J. Huizenga
Prof. Dr. Ir. M. de Bruin
Dr. Ir. A.G. Denkova
Dr. ir. D.R. Schaart

Committee members: Dr. ir. D.R. Schaart
Dr. ir. P. Bode
Dr. K. Djanashvili

Date of submission: 22-07-2015 Date of presentation: 29-07-2015
2
Abstract

Polymersomes, polymeric nano-carriers composed of amphiphilic block copolymers, are promising
candidates for transporting (radio)pharmaceuticals to tumour cells. While methods have been
developed for trapping both diagnostic and therapeutic amounts of radionuclides in these
polymersomes, there is not much known about the loading process and the state of the radionuclide
in the polymersomes. At this point perturbed angular correlation (PAC) spectroscopy becomes
essential, as it is a useful method for gaining information about the chemical surroundings and
physical structures of different gamma-emitting radioisotopes.

PAC is a gamma ray spectroscopy technique which can be used for the investigation of hyperfine
interactions. A hyperfine interaction is the interaction between a nucleus and its environment.
Hyperfine interactions arise in the intermediate state of a decaying radionuclide. This decay is then
perturbed with a time-dependent factor and can be measured from the angular correlation function.
The angular correlation function is expressed as the number of coincidences per unit time as function
of angle. The number of coincidences is measured using the PAC spectroscopy.

The polymersomes are composed of poly(butadiene-b-ethylene oxide) block copolymers, and the
labelling has been achieved by transportation of the radionuclide, complexed to a lipophilic ligand,
through the hydrophobic bilayer into the aqueous cavity containing 0.5 M of KH PO . A sufficient
2 4
amount of the radionuclides was encapsulated in the polymersomes (>90 % loading efficiency).
Subsequent to the loading, the sample was immersed in liquid nitrogen, after it was counted on the
PAC setup for 6 hours at 90 and 180 angles.

To confirm a working PAC setup, measurements on directional correlation and a simple perturbed
system are performed. Excellent agreement with literature has been observed for cobalt-60
directional correlation measurements (G22 of 0.1016 vs 0.1020 in [18]). The hafnium metal
measurements, which only undergo a static interaction, are compared with literature [39]. The
results give an indication of a reliable PAC setup.

Final measurements are performed with indium-111. The results obtained for the loaded
radionuclides in polymersomes indicate that differences could be distinguished. The results indicate
that the radionuclides will move from the bilayer towards the core of the polymersome. There was
no clear bonding observed between the chelator and the radionuclides. The used side of the
polymersomes (100 nm, 800 nm) are expected not to influence the measurement.

3
4
Contents

Abstract ................................................................................................................................................... 3
Abbreviations and acronyms ................................................................................................................... 7
1 Introduction ..................................................................................................................................... 9
2 Polymersomes ............................................................................................................................... 11
3 Theory of PAC ................................................................................................................................ 13
3.1 Background on PAC ............................................................................................................... 13
3.2 γ-γ cascade ............................................................................................................................ 13
3.3 Hyperfine interactions ........................................................................................................... 14
3.3.1 Magnetic dipole interaction .......................................................................................... 14
3.3.2 Electric quadrupole interaction ..................................................................................... 15
3.4 Angular correlation of γ-γ cascade ........................................................................................ 16
3.5 Perturbed angular correlation of γ-γ cascade ....................................................................... 17
3.5.1 Unperturbed interaction ............................................................................................... 19
3.5.2 Static interaction ........................................................................................................... 19
3.5.3 Time dependent interaction .......................................................................................... 20
4 Experimental PAC setup ................................................................................................................ 23
4.1 Detectors ............................................................................................................................... 24
4.1.1 Scintillator ...................................................................................................................... 24
4.1.2 Photomultiplier tube ..................................................................................................... 25
4.2 Slow coincidence circuit ........................................................................................................ 25
4.2.1 Spectroscopy amplifier .................................................................................................. 25
4.2.2 Coincidence unit ............................................................................................................ 26
4.3 Fast coincidence circuit ......................................................................................................... 26
4.3.1 Constant-fraction Discriminator .................................................................................... 26
4.3.2 Time to amplitude converter ........................................................................................ 28
4.4 The output signal ................................................................................................................... 28
4.5 Signal processing ................................................................................................................... 29
5 Materials ........................................................................................................................................ 31
5.1 Radionuclides ........................................................................................................................ 31
5.2 Polymersome preparation and loading (with indium-111) ................................................... 31
5.3 Equipment ............................................................................................................................. 32
5.3.1 Electronic components .................................................................................................. 32
5.3.2 Sample holder ................................................................................................................ 32
5
5.3.3 Sample encapsulation ................................................................................................... 33
6 Methods ........................................................................................................................................ 35
6.1 Characterization of the PAC setup ........................................................................................ 35
6.1.1 Energy- and time resolution measurements ................................................................. 35
6.1.2 Detector performance ................................................................................................... 36
6.1.3 Limitations of the setup................................................................................................. 37
6.2 Directional correlation measurements ................................................................................. 37
6.3 Hafnium-181 measurements ................................................................................................. 38
6.4 Indium-111 measurements ................................................................................................... 38
7 Results and discussion ................................................................................................................... 41
7.1 Characterization of the PAC setup ........................................................................................ 41
7.1.1 Time resolution.............................................................................................................. 41
7.1.2 Energy resolution .......................................................................................................... 42
7.1.3 Detector performance ................................................................................................... 43
7.1.4 Limitations of the setup................................................................................................. 46
7.2 Directional correlation measurements ................................................................................. 48
7.3 Hafnium-181 measurements ................................................................................................. 50
7.4 Indium-111 measurements ................................................................................................... 53
8 Conclusion ..................................................................................................................................... 59
9 Outlook .......................................................................................................................................... 61
10 References ..................................................................................................................................... 63
11 Appendices .................................................................................................................................... 67
12 Poster ............................................................................................................................................ 77
Acknowledgements ............................................................................................................................... 79

6
Abbreviations and acronyms

Abbreviation Explanation
PAC Perturbed angular correlation
PMT Photomultiplier tube
LaBr (Ce) Lanthanum bromide
3
ADC Analog to digital converter
MCA Multi channel analyser
CFD Constant fraction discriminator
TAC Time to amplitude converter
SBD Radiation protection services
Hf Hafnium
In Indium
Na Sodium
Cs Caesium
Co Cobalt
LN Liquid nitrogen
2

7
8
1 Introduction

Cancer is one of the leading causes of morbidity and mortality worldwide. In 2012, approximately 14
million new cases and 8.2 million cancer-related deaths were recorded. It is expected that cancer
cases will rise from 14 million in 2012 to 22 million within the next 2 decades. [1] These numbers
indicate that knowledge about the causes of cancer and interventions to prevent and manage the
disease are essential. Every cancer type requires a specific treatment, encompassing one or more
modalities, such as surgery, chemotherapy and radiotherapy. Although surgery is considered to be
one of the most effective ways to treat cancer patients, in cases where the disease metastasizes
radionuclide therapy is, next to chemotherapy, one of the only proven alternatives. Radionuclides
are, therefore, indispensable in both diagnostics as in therapy. [2]

In radiotherapy, tumours are irradiated to kill the tumour cell. This can be done in two different
manners: from outside the body (external radiotherapy) and inside the body (internal radiotherapy).
During external radiotherapy, the tumour is irradiated using several beams. A disadvantage is that
surrounding healthy tissue will receive a dose as well. Using internal radiotherapy, the radiation
could be delivered very locally. Hence, this method destroys the tumour cells while simultaneously
preserving the surrounding healthy tissue as much as possible. Another advantage of internal
radiotherapy is that small metastases can be treated.

Using internal radiotherapy, radionuclides will be injected into the body. Preventing the
radionuclides to spread throughout the body, radiation can be loaded into a carrier. One of these
carriers which are investigated are polymersomes. Polymersomes are polymeric nano-carriers which
can be used to transport therapeutic or diagnostic amounts of radionuclides to specific sites in the
body. [3] Polymersomes can be loaded with alpha-, beta- or gamma-emitting radionuclides, this
process is called radiolabelling. Once the polymersomes are loaded, the radionuclides are
transported to the tumour site. Ionizing radiation is emitted by the radionuclides in the
polymersomes. They could kill tumour specific cells and limit the damage to healthy surrounded
tissue. [4,5]

There is not much known about the specifics of radiolabelling polymersomes. Once the radionuclides
are loaded, is the radiation inside the core or in the bilayer of the polymersomes? In what state is the
radionuclide inside the polymersome? Perhaps these and similar questions could be answered using
perturbed angular correlation (PAC) spectroscopy.

PAC spectroscopy is a very useful method to gain information about the chemical surroundings and
physical structures of different radioisotopes. PAC relies on measuring the variations in time between
two decays as a function of time and angle. These variations are caused by the surroundings (solid,
liquid etc.) of the radionuclide and PAC is able to measure these.

PAC is a non-invasive method, hence no limitations in the choice of sample environment are present.
PAC experiments can be carried out with samples in any aggregate state and in a wide range of
pressures, temperatures and external electromagnetic fields. [5] Only minute quantities of biological
material (10-12 mol) are needed to perform PAC experiments. [6]

The theory behind PAC spectroscopy is based on the decay of an excited state of a decaying nucleus
to a metastable state, by the emission of a first radiant. This emission is followed by a decay from the
metastable state to the ground state where a second radiant is emitted. [7] This metastable state is
called the intermediate level of the cascade.

9
Radionuclides used for PAC spectroscopy should have specific properties:
• the nuclides should have a nuclear de-excitation through a γ-γ cascade
• the intermediate level of the γ-γ cascade should have a spin ≥ 1
• the half-life of the intermediate state should be between 2 ns and 1 µs

It is noted that a useful range of the half-life of the intermediate state is not well defined. When the
intermediate life-time is small, the system becomes unperturbed, so the system is not able to
measure any interactions. Unfortunately, the exact point from where interactions could be measured
has not been found in literature.

Two radionuclides which have favourable properties and are therefore used in typical PAC
experiments are indium-111 and hafnium-181. The activity of the samples is typically between 1-100
µCi.

The ultimate goal of research in this field is to measure radionuclides encapsulated in polymersomes
in vivo. In this manner, patients can be treated in an effective manner by local radiotherapy and
while using PAC spectroscopy important information about the treatment and diagnostics are
gathered. The first goal in this project is to investigate the possibility of using PAC can be used in vitro
experiments. When these results are positive, further research can be performed to explore the
possibilities of using PAC in vivo.

The objective of this thesis is to determine the state of radionuclides inside polymersomes using
perturbed angular correlation spectroscopy. To answer this question, a properly working PAC
spectroscopy setup is necessary. At the beginning of this project, an old setup was available and
explored to investigate if it was still working. Confirmation of a properly working PAC setup should be
done by several experiments. The time and energy resolution and other setup limitations are
important quantities to investigate. After this, directional correlation measurements are performed
with an unperturbed radionuclide, which gives a first clue about the working of the setup. Hafnium-
181 metal only shows static interactions and has a short half-life of the intermediate state. Once
these measurements are preformed, first preliminary measurements with indium-111 in different
states and loaded into polymersomes are performed. These measurements are performed to confirm
the hypotheses that PAC is a suitable method to use in in vivo measurements.

In this thesis different topics are covered in order to answer the research question. First, the working
principle of polymersomes is described, followed by a brief historical outline and a quick overview of
the working of PAC. Further, knowledge about hyperfine interactions, angular correlation and
perturbed angular correlation is necessary, which will be explained in section 3.3 – 3.5. Then, the PAC
setup will be declared in more detail. In a subsequent material section, a list of all the used materials
and electronics can be found. The experiments done are explained in the method section. From this
the results obtained in this research are described and discussed. The last part of this thesis contains
the conclusions and an outlook for further research.
10