SCHOOL OF CHEMISTRY 2012 - 2013
MChem – Year Four - Module CH3401 (60 credits)
FINAL YEAR PROJECT THESIS
The Preparation and Evaluation of Novel Polymeric MRI Contrast Agents
Darren Gullen
1
Acknowledgements
I wish to acknowledge Dr Angelo Amoroso and Dr Alison Paul for their support and
assistance throughout the duration of the project, without them I would be lost and this project
would not have been possible. I would also like to thank the members of the Inorganic Chemistry
research group and the Soft Matter research group for their help and contributions.
2
Contents
Abstract....................................................................................................................................4
1. Introduction..........................................................................................................................5
1.1 Aims...………………………………………………………………………………………….......................6
1.2 Principles of NMR/MRI..........................................................................................7
1.3 Relaxivity Processes...............................................................................................9
1.4 Fast Field Cycling Relaxometry (FFC)....................................................................16
1.5 Relaxivity ...…………………………………....……………………………………….……….……….……..19
1.6 Contrast agents.....................................................................................................26
1.7 Chitosan...…………………………………....……………………………………….............................31
2. Experimental........................................................................................................................33
2.1 Materials...............................................................................................................33
2.2 Instrumentation and Methods..............................................................................33
2.3 Preparation of the chitosan control groups….......................................................34
2.4 Relaxivity measurements……………………………….……………………………......................35
3. Results and discussion.........................................................................................................36
3.1 Synthesis of the modified chitosan polymers ……………..…………………...................36
3.2 FT-IR data of DTPA-Ch, EDTA-Ch and Chitosan.....................................................37
3.4 NMR data of DTPA-Ch, EDTA-Ch and Chitosan.....................................................38
3.5 Relaxivity Data...................................................................................................... 39
4. Conclusions..........................................................................................................................47
Appendix...…………………………………………………………………………………….…..…..............................49
References...............................................................................................................................59
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Abstract
Firstly the polymeric contrast agent [Ch-DTPA-Mn] was synthesized and studied as a
potential Magnetic Resonance Imaging (MRI) contrast agent. Secondly control groups were
established which consisted of physical mixtures of (Ch-Gd3+) and (Ch-Mn2+). These control groups
helped to determine that there was no coordination of chitosan to the paramagnetic species in
solution, so percentage loadings of the paramagnetic species to the chelating ligands could be
accurately determined from relaxivity data. [Ch-DTPA-Mn] was synthesised successfully by a cross
coupling reaction through which an amide bond was formed between the chelating ligand (DTPA)
and chitosan. The complexes were characterised using IR spectroscopy, NMR Spectroscopy,
relaxometry and SANS. The r1 relaxivity of this contrast agent was obtained and compared to the first
FDA approved contrast agent Magnevist ([Gd-DTPA], 3.4-4.5 mM-1s-1, 1.5T, 25oC). The r1 relaxivity of
this [Ch-DTPA-Mn] was found to be 3.85 mM-1s-1 (30 MHz and 25°) which is lower than that of
Magnevist. However the increase in relaxivity to 20.08 mM-1s-1 (10 kHz and 25°C) of this complex at
low field strengths is a promising property of Manganese based contrast agents. It was also found
that 1mM Ch-DTPA fully saturated has bound ~ 0.18-0.195 mM Mn2+, which under the assumption of
a 1:1 ratio of metal to chelating ligand, this corresponds to an 18-19.5% loading of DTPA on chitosan.
SANS data revealed that the shape of the curves are very similar for all three solutions of Chitosan,
[Ch-DTPA] and [Ch-EDTA], which suggests they all possess similar conformations in solution,
however the slight differences in gradients between the three complexes suggest that they vary
slightly in size which is a good indication that they differ structurally. The relative relaxivity of [Ch-
DTPA-Mn] was also compared to some polymeric contrast agents previously studied by Rana. Who
found that effectiveness of these polymeric contrast agents to the increase in the relaxation rates of
water protons local to them were; [Ch-DTPA-Gd] > [Ch-EDTA-Mn] > [Ch-EDTA-Gd] > [Gd-DTPA]1 On
comparison of the relaxivities [Ch-DTPA-Mn] had the lowest relaxivity which was to be expected due
to the lack of water molecules directly coordinated to the metal. These results show that certain
SBM theory parameters, such as the hydration number have a bigger effect on the relaxivity than
other parameters such as the rotational correlation time of the complex.
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1. Introduction
Magnetic Resonance Imaging (MRI) is used to visualize detailed internal structures of the
body and has expanded and continuously developed as an imaging technique in diagnostic medicine.
There have been vast improvements and changes in the area of MRI especially in the study and
synthesis of new contrast agents that display higher relaxivities thus enhancing the visibility of
internal body structures within an MRI image. MRI is very useful for imaging many areas of the body
such as the brain, the heart, inflammation sites and even tumours from cancer because it has a high
contrast resolution which allows for easy differentiation of various soft tissues. MRI images can also
track the flow of blood throughout the body via intravenous injections of contrast agents; so it can
highlight problems with circulation, such as blockages or ruptures.
An MRI image is made by making use of the property of nuclear magnetic resonance of the
hydrogen nuclei inside the body. The contrast observed in the MRI image is because of differences in
relaxation rates of the water protons between various tissues. This is extremely useful considering
that the human body is made up of approximately 63% hydrogen atoms by atomic percent from fat
and water.2 MRI is preferred over other imaging techniques, such as CT and fluorescence microscopy
because it is non-invasive and uses no ionising radiation. This means it is safe to use in patients who
may be vulnerable to the effects of radiation. Another useful aspect of MRI is that any part of the
body can be analysed at any angle or orientation without the patient ever having to move. MRI can
also produce 3-D images by stacking together all of the 2-D images; this gives a full spatial
reconstruction of the area being scanned and in some cases a full body image can be produced.
Contrast agents improve the contrast resolution of an MRI image by increasing the ability to
differentiate between normal and diseased tissue; so it enhances the appearance of soft tissue such
as muscles, the brain and the heart. Commercially used contrast agents typically consist of low
molecular weight organic ligands bound to highly paramagnetic metal centres, such as [Gd-DTPA]
(known commercially as Magnevist). New research in this field is in the development of reagents
designed to bind to protein receptors at the cell surface, leading to targeted studies (e.g. targeted
uptake of the contrast agent to a specific organ of the body). The performance of currently existing
contrast agents are limited by a relatively low local concentration of the contrast agent at the
desired site, so a higher contrast agent relaxivity is required to compensate for the low receptor
concentrations. Theoretical studies suggest that extremely high relaxivities are possible if all
appropriate parameters are optimised. One such parameter is the tumbling rate of the molecule;
increasing the molecular weight of the organic section through the use of polymers increases the
relaxivity. This project will focus around a Manganese based contrast agent, which is coordinated to
the modified polymer, Chitosan-DTPA, via the chelating ligand.
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1.1 Aims
This report is extended research into a project initiated by Banhu Partap Singh Rana, who
synthesised and analysed three novel polymeric contrast agents;[Ch-DTPA-Gd], [Ch-EDTA-Gd] and
[Ch-EDTA-Mn]which had relaxivity values, r1, of 7.97, 7.45 and 5.83 mM-1s-1 (at 30MHz and 25°C) and
percentage loadings of the ligands onto Chitosan of 24%, 19% and 30% respectively.1 However the
relaxivity of [Mn-DTPA-Ch] was unable to be obtained by this undergraduate due to the difficulty in
keeping the Ch-DTPA complex from precipitating out of solution whilst relaxivity measurements
were being undertaken.
The aims of this project were to synthesis the final polymeric contrast agent and obtain its
relaxivity data to complete the matrix of systems investigated by Rana. however the polymeric
solution would need to be made slightly more acidic to stop the precipitation of the polymer. To
complete the study critical control experiments were required; which consisted of [Chitosan-Gd] and
[Chitosan-Mn] solutions being prepared and their relaxivity data obtained to determine whether
Chitosan coordinates to either Gadolinium or Manganese. Finally the solutions of Chitosan, [Ch-
DTPA] and [Ch-EDTA] would be synthesised and their respective reduced format SANS data would be
obtained to compare the relative sizes and shapes of these polymer complexes in solution.
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1.2 Principles of NMR/MRI
All particles such as electrons, protons and neutrons possess a spin. In many atoms the
nucleus has no overall spin related to it due to the balance of spin vectors between the neutrons and
protons; however in some cases such as a 1H nucleus there is no neutron present to cancel out the
spin of the single proton and so it possesses an overall nuclear spin. This spin is a fixed characteristic
property of a nucleus and is denoted by the spin quantum number, I, and can either be an integer or
half integer. A sample possessing a net spin can absorb a photon of frequency, ν, when exposed to
an external magnetic field of strength B, and this frequency depends on the gyromagnetic ratio, γ, of
the particle. For hydrogen, γ = 42.58 MHz / T and I= ½
ν = γB (1)
When an external magnetic field is applied to a 1H nuclei it splits into two spin states; a
higher energy spin state, β, where the proton spin aligns anti-parallel to the external magnetic field
(mI = -½ )and a lower energy spin state, α, where the proton spin aligns parallel to the magnetic field
(mI = +½). A particle can undergo a transition from the lower energy state to the higher energy state
if it absorbs a photon of energy which matches exactly that of the energy gap between the two spin
states as shown in figure 1. The energy, E, of a photon is related to its frequency, ν, and Planck’s
constant, h shown by equation (2).
E = hν (2)
For MRI the frequency (ν) of the absorbed photon is called the resonance frequency or the
Larmor frequency. The frequency is typically between 15 and 80 MHz when imaging hydrogen in
clinical MRI.
Through the combination of equations (1) and (2) a new equation (3) can be produced which
shows the energy required by a photon to cause a transition between the α and β spin states. When
the energy of the photon exactly matches that of the energy gap between the lower and higher
energy spin states, an absorption of energy occurs.3
E = ħγB (3)
7
Energy
0
No Field Applied magnetic fieldβ
α
E = ħγB
mI = +½
mI = -½
Figure 1. Nuclear spin energy levels of spin-½ nucleus with positive magnetogyric ratio (1H)
Through the use of Boltzmann statistics described by equation (4), we can determine the
population of spins in the α and β spin states.
N-/N+ = e-E/kT (4)
Where E is the energy difference between the higher and lower energy spin states, k is
Boltzmann's constant, and T is the temperature in Kelvin. At room temperature the population of
spins in the α state, N+, is slightly higher than the population of spins in the β state, N-. As the
temperature is increased the ratio N-/N+ approaches one, but if the temperature is decreased then
the ratio N-/N+ also decreases, which increases the intensity
An MRI signal is a result of the difference in energy between that absorbed by spins which
are transitioning from the lower energy α state to the higher energy β state and that emitted by the
spins which are simultaneously transitioning from the higher energy β state to the lower energy α
state. This means that the signal intensity is directly proportional to the population difference of the
states. MRI has become a useful and widely used imaging technique due to its ability to detect these
subtle population differences. MRI is a highly sensitive technique and this is due to resonance at a
precise frequency between the spins of the H nuclei and the spectrometer. However the natural and
biological abundance of the isotope being targeted influences the signal intensity. The 1H isotope
has a natural abundance of 99.985%4 and a biological abundance of 63%2 which is useful because
MRI focuses on the 1H nuclei.
8
z
y x
M0
B0
1.3 Relaxivity Processes
Relaxivity processes and spins function on a microscopic scale (e.g. individual spins);
however it is too complicated to describe spins on a microscopic scale and so it is normally described
on a macroscopic scale. This means a group of spins ‘feeling’ the same magnetic field strength can be
denoted as a spin packet and the magnetic field created through the spins in each spin packet can be
denoted by a magnetisation vector. The magnitude of each magnetisation vector is directly
comparative to (N+ - N-). The net magnetisation is the vector sum of all magnetisation vectors and at
equilibrium it is known as the equilibrium magnetisation, M0, shown in figure 2. Here it lies along the
Z axis, parallel with the direction of the applied magnetic field, B0. In this conformation, the Z
element of magnetisation, referred to as the longitudinal magnetisation, MZ, is equal to the
equilibrium magnetisation, M0. Also there are no transverse elements (MX or MY) of magnetisation
here.
Figure 2. Net Magnetisation at
equilibrium
1.3.1 T1 Process (Spin-Lattice
Relaxation)
The spin-lattice (or longitudinal) relaxation
time, T1, is a quantification of the rate of transfer of energy
from the nuclear spin system to the neighbouring
molecules in the lattice. After a spin system has been
exposed to a radio frequency (RF) radiation of energy, which is equal to that of the energy difference
between the α and β spin states, the spin system becomes saturated and the net magnetisation, Mz
equals zero. When this occurs the net magnetisation vector is composed of transverse elements.
This occurs because some of the +½ nuclei are excited to the -½ state. When the net magnetisation is
in the XY plane, it revolves around the Z axis at the Larmor frequency. This is known as precession
which becomes coherent about the z-axis. The net magnetisation then returns to equilibrium in a
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M0 = Mz
Saturation with RF radiation T1
Mz = 0 Mz = 0.63 M0
process called relaxation. The transverse magnetisation vector of this spin group starts to de-phase
and the coherency disappears, the population of the +½ state increases and energy is released which
can be detected by a receiver. The net magnetization spirals back into the longitudinal vector and
eventually the equilibrium state is re-established. (Figure 3)
Figure 3. Change in net magnetisation with T1 relaxation
The spin lattice relaxation time or longitudinal relaxation time, T1, is the time constant that
describes how MZ returns to its equilibrium value. Equation (5) administers this behaviour as a
function of the time, t, after the displacement of MZ.
Mz = Mo (1 - e-t/T1) (5)
So T1 is an exponential process and is the time to decrease the change amongst the
longitudinal magnetization, MZ, and its equilibrium value, M0, by a factor of e. After time T1, the
longitudinal magnetisation will have returned to 63% of its equilibrium value. (Figure 4)
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M0 = Mz
180° T1 90°
180°
T1
90°
Detection
Figure 4. A graphical representation of equation 5, showing how T1 is an Exponential process.
The standard method for recording T1 of a sample is known as an inversion-recovery pulse
sequence. Firstly, a 180° RF pulse is applied across the sample. This inverts the net magnetization of
the protons along the -Z axis. A time period, τ, is allowed, during which spin-lattice relaxation occurs
Causing the Mz vector to return back toward its equilibrium position along the +Z axis. However
before it reaches its equilibrium point, a 90° RF pulse is applied across the sample causing the
longitudinal magnetization to rotate into the XY plane. Precession in the XY plane of the
magnetization occurs about the Z axis and de-phases to give free induction decay (FID) which can be
detected by an RF Coil. The inversion time, TI, is considered to be the time between the initial 180°
pulse and the 90° pulse. (Figure 5)
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T1
Figure 5. Change in magnetisation vectors during an Inversion recovery pulse sequence
Different tissue types have different T1 values, which mean that some tissue signals, such as
those from fat, can be suppressed. In a fat suppressed image only the water from the sample is
imaged.5 A method for distinguishing between different tissue types is known as a short T1 inversion
recovery pulse sequence (STIR). In this technique the second pulse of RF radiation in the inversion
recovery pulse sequence is applied to the sample at a specific time according to its value of T1. This is
useful because the value of T1 in fat is much lower than that of water, therefore if the second pulse
of RF radiation is applied at the exact time that the net magnetisation of the fat is equal to zero, then
the fat signal will be suppressed and only the water signals from the tissues will be detected. This
allows for a much clearer contrast of the images to be obtained which is highly advantageous when
running diagnostic tests on patients using MRI. (Figure 6)
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Saturation with RF radiation T2
t = 0
Figure 6. Suppression of fat signal by STIR
1.3.2 T2 Process (Spin-Spin Relaxation)
The spin–spin, or transverse relaxation time, T2, occurs simultaneously alongside T1. It is a
quantification of the rate of the decay of the magnetization vector within the xy plane, and can be
considered as the time constant that describes how the transverse magnetization, MXY, returns to
equilibrium. The transverse magnetisation, MXY, is perpendicular to the applied external magnetic
field, B0. In most cases T2 is less than or equal to T1 but it rarely exceeds T1, this is because re-
establishment of the magnetization vector to the z-direction inherently causes loss of magnetization
in the xy plane. (Figure 7)
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M0 = Mz
180°
Water
Fat
T1 90°
No longitudinal magnetisation from fat
Detection of water from signal in xy plane
T2
Figure 7.
Change in net
magnetisation
associated with T2
relaxation
Equation (6) administers this behaviour as a function of the time, t, after the decay of Mxy.
MXY is the transverse magnetisation after time, t and MXY0 is the transverse magnetisation at
equilibrium. Figure 6 shows how T2 is an exponential process; after time T2, the Transverse
magnetisation will have returned to 37% of its equilibrium value. (Figure 8)
MXY =MXYo e-t/T2 (6)
Figure 8. A graphical representation of equation (6), showing how T2 is an exponential process.
There are two factors which influence the de-phasing of the transverse magnetisation. The
first factor is due to the fact that every spin packet that makes up the net magnetisation has its own
slightly different magnetic field and this slight difference in magnetic fields interact with one another
and cause each spin packet to have its own Larmor frequency. It is noted that the amount of phase
difference rises with time. These molecular interactions lead to a ‘pure’ T2 molecular effect. The
second factor is a result of the external magnetic field (B0) being subjected to each spin packet
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M0 = Mz
180°
t90°
Re-phasing of the spins Detection
180°
t
90°
Detectiont
t
De-phasing of the spins
differently. This is because of the numerous proton environments in the samples and is known as the
‘inhomogeneous’ T2 effect. When the ‘pure’ and ‘inhomogeneous’ time constants are combined it
can be labelled as T2*, which is shown by equation (7).
1/T2* = 1/T2 (pure) + 1/T2 (inhomogeneous) (7)
T2 can be equal to or less than T1 but it can never exceed T1, this is because re-establishment
of the magnetization vector to the z-direction inherently causes loss of magnetization in the xy
plane.
A T2 relaxation of a sample can be measured using a spin-echo pulse sequence and is
commonly used in MRI.6, 7 A spin-echo pulse sequence is very similar to an inversion recovery pulse
sequence except a 90° RF pulse is applied first across the sample causing the longitudinal
magnetization to rotate into the XY plane. At this point the transverse magnetization starts to de-
phase. After a time, t, a second pulse of 180° RF is applied and this second pulse inverts the
magnetisation vectors and causes the spins to re-phase. The spins then re-phase to produce a signal
called an echo and this is detected at time 2t. (Figure 9)
15
Figure 9. Change in magnetisation vectors during a spin-echo pulse sequence
1.4 Fast Field Cycling (FFC) Relaxometry
FFC relaxometry is a technique used to quantify spin-lattice and spin-spin relaxation rates, r1
and r2, at numerous field strengths. FFC relaxometry allows analysis at a number of frequencies
through the use of the same instrument. This is a great advantage because it doesn’t require the use
of multiple NMR machines; each tuned to different frequencies. It is known as “Fast Field Cycling”
relaxometry because the time it takes to switch between different fields is considerably shorter than
the sample’s longitudinal relaxation time, T1. During the process the relaxometer goes through a
range of different external magnetic field strengths, normally ranging from 10kHz-30MHz, and It
measures the spin-lattice relaxation time of a sample at each unique field strength. This produces a
Nuclear Magnetic Resonance Dispersion (NMRD) profile, which can be analysed to obtain the
relaxivity of a sample. These NMRD profiles can be gathered via two different methods; a pre-
polarised sequence (PPS), and a non-polarised sequence (NPS).
1.4.1 Pre polarised sequence technique (PPS)
The PPS technique works by applying a strong magnetic field (Bp), which increases a sample’s
nuclear magnetisation. The strong magnetic field polarises the spins of the sample after which the
magnetic field is changed to a lower strength, Br. The sample is then left for a set period of time, τ, to
allow for relaxation processes to occur. The magnetisation of the sample in this time period is
16
B0
0 t
Polarization of spins DetectionRelaxation
Bp
Br
Bd
τ
tRF
FID
parallel to the externally applied magnetic field and its magnetisation value is initially equal to the
equilibrium magnetisation, MZ(0), as shown by equation (9).
M0 = MZ(0) = M0(Bp) (9)
The sample relaxes towards its new vector, M0Br, which is shown in equation (10).
Mz(τ) = M0(Br) + [M0(Bp) - M0(Br)] е(-τ/ T1Br) (10)
After this process, the magnetic field is switched to a higher flux density field of strength, Bd.
The sample is then subjected to a 90° pulse of RF radiation, after which the signal is detected
through FID. However the field needs to be reverted back to Bp before the process can be repeated;
this is done to switch the net magnetisation vector to its original state. The PPS is a useful technique
if the relaxation field strength is much lower than the polarisation field strength. (Figure 10)
Figure 10. A graphical representation of the difference in magnetic field strength during a pre-
polarised sequence
17
1.4.2 Non Polarised Sequence technique (NPS)
The relaxation and detection periods of a NPS and PPS are identical; the only thing that
differs is the sample magnetization at the beginning of the relaxtion period. For a NPS there is no
initial polarization of the spins before relaxation therefore the relaxation curve is created from a
standard inversion recovery method in which a 180° pulse rotates the net magnetisation vector
down into the -Z axis. After that the net magnetisation experiences a spin-lattice relaxation and the
net magnetisation vector returns to its equilibrium value along the +Z axis as shown in equation (11)
Mz(τ)detected = Mz
∞ - ΔMz
eff e(−τT 1Br ) (11)
After this process the magnetic field is switched to the field strength, Bd, and the sample is
then subjected to a 90° pulse of RF radiation after which the signal is detected through FID. A NPS is
used when the relaxation field strength approaches that of the polarisation field strength.(Figure 11)
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B0
0 t
Preparation Detection
Relaxation
Br
Bd
τ
tRF
FID
Figure 11. A graphical representation of the difference in magnetic field strength during a
non-polarised sequence
1.5 Relaxivity
There have been many great advancements in the production and use of contrast agents
used for MRI. Gadolinium based contrast agents are by far the most used and widely available
contrast media for MRI. However there are other medically approved contrast agents based on
Manganese, iron oxide and iron platinum nano particles. Contrast agents are able to increase the
contrast of an MRI image by influencing and enhancing the relaxation time of protons local to the
paramagnetic species. The magnitude of this effect is measured as relaxivity, r1 or r2, which has units
19
of mM-1s-1 and is the change in 1/T1 or 1/T2, respectively, of the contrast agent at 1mM concentration
at a specific magnetic field strength and temperature. (Δ1/T1) is the change in relaxation rate of a
sample after adding a contrast agent containing a metal concentration [M], shown by equation (12)
r1 = (Δ1/T1) / [M] (12)
As shown from equation (13) the 1/T1 value of water is represented by the 1/T1 intercept of
the line at zero concentration of Gd3+ and the relaxivity, r1, can be determined from the gradient of
the line on the graph 1/T1 of water against the concentration of gadolinium Gd3+, which gives a linear
relationship.
1/T1 (Measured) = 1/T1 (Water) + r1 [Gd3+] (13)
Pulse sequences are split into two types, T1-weighted or T2-weighted; the sequences that
highlight changes in 1/T1 are known as T1-weighted and those that highlight changes in 1/T2 are T2-
weighted. But Contrast agents based on gadolinium increase both 1/T1 and 1/T2 by approximately
the same amount. Despite this, these contrast agents are more visible using T1-weighted scans
because the percentage difference in 1/T1 in the tissue is higher than the percentage difference in
1/T2.8
The relaxation times are used to assess the efficiency of the agents.8, 9, 10,11 However most of
the contrast agents that are used in modern medicine and MRI scans are extracellular, non-specific
and less effective than theorised.12 This means high concentrations are needed local to the area
being scanned to achieve enough contrast on a MR image.13, 14
1.5.1 Factors affecting T1 Relaxivity
The mechanism of relaxivity can be separated into three types of water molecules as shown
in Figure 12; those that are directly coordinated to the paramagnetic metal centre are known as
inner sphere water molecules, those that are involved in the hydration of the metal complex and are
known as second sphere water molecules and finally outer sphere water molecules are those that
reside in the bulk solution. It is the overall combination of these three types of water molecules that
contribute to the overall relaxivity of the complex. These three types of water molecules can be split
20
i = 1 or 2
Metal
Ion
Outer SphereSecond Sphere
Inner Sphere
H
O
H
H2
OH2
OH2
O
H2
OH2
O
H2
O
H2
O
H2
O
H2
O
H2
O
H2
O
H2
O
H2
O
H2
O
H2
O
HO
H
into two mechanistic categories; the inner sphere mechanism and the outer sphere mechanism.
Equation (14) shows how they contribute to the overall relaxivity.
( 1T i )overall=( 1T i )inner+( 1T i )outer (14)
Figure 12. Sketch of the inner, second and outer sphere water molecules
There are several parameters that can affect the relaxivity of these water protons in the
presence of a Gd3+ contrast agent.15 The Solomon–Bloembergen–Morgan (SBM) theory and the
generalised SBM theory (GSBM)9, 16 state that the overall relaxivity is associated to a group of
physico-chemical parameters. These parameters are obtainable via NMR examinations based on
SBM theory and are frequently used to characterise contrast agents. Equation (15) shows the
primary factors that affect the overall relaxivity of a contrast agent under ambient conditions.
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r1 = C . q . μeff2 . τc . r-6 (15)
Where C is a constant, q is the number of water molecules directly coordinated to the metal
centre in the inner sphere (hydration number), μeff is the effective magnetic moment, τc is the
molecular correlation time (molecular tumbling rate) and r is the Gd---H (H2O) bond distance.
However the molecular correlation time (τc), is defined by the following parameters; the
rotational correlation time (τr), the electronic correlation time (τs), and the proton residence time
(τm),reciprocal of the water exchange rate. Equation (16) shows how these terms are associated
together to give an expression for τc.
τc -1 = τr -1 + τs -1 + τm -1 (16)
The most significant parameters for the designing of the polymeric contrast agent are those
which can be controlled chemically.15 These parameters are ordered depending on the significance in
roles that they play for the enhancement of relaxivity (Figure 13):
1. q – The hydration number
2. τm - The residence time of the coordinated water molecule
3. τr - The rotational correlation time
4. qss and τm ss – Second and outer sphere hydration number and mean residence time
respectively
22
Figure 13. Parameters of inner sphere relaxivity
The hydration number (q) has considerable impact on the inner sphere relaxivity; this is
because the water molecules directly coordinated to the paramagnetic species are part of the inner
sphere. The hydration number needs to be large enough to have an increase in relaxation, however,
the stability of the complex would be reduced if this value was too large which would increase the
chance of toxic metal ions being released in the body. X-ray structure analysis is a technique that can
determine the number of water molecules directly coordinated to the paramagnetic species in the
inner sphere. However it should be noted that this solid state structure does not always correspond
to that of the structure present in solution. But there are also other techniques available to
determine the hydration number of the contrast agent in solution such as, Lanthanide Induced Shift
NMR,17,18,19 EPR spectroscopy,20, 21and luminescence,22, 23, 24
23
9
Another parameter that increases the overall relaxivity of a macromolecular contrast agent
at 1.5T is a short residence time, τm around 10ns. The 1.5T magnetic field is the most common
magnetic field used in modern MRI scanners.15, 25 At higher fields, such as those in the most advanced
MRI scanners, contrast agents with even shorter τm values between 1-10ns would be needed.26, 27 The
faster the water exchange rate and hence an optimally short water residence time, the better the
relaxation, although this can be limited by steric hindrance. The τm term is mainly affected by the
water exchange mechanism, the charge of the complex, steric restrictions about the water-binding
site and the accessibility of the solvent.
The rotational correlation time, τr, represents the molecular tumbling rate of a complex. At a
magnetic field strength of 1.5T, a minimum time of 10ns would be an ideal τr value.27 Increasing the
rotational correlation time which gives an optimally slow rate of molecular tumbling can result in a
vast enhancement in relaxation; as long as the ligand is designed so that the water exchange rate is
not so slow that it hinders the gains from the increase in τr.9, 28, 29, 30 This enhancement in relaxation
can be obtained by binding the paramagnetic species to a macromolecule via a number of different
methods to increase the effective molecular volume and therefore give a slower but more optimal
molecular tumbling rate. The macromolecules chelated to the paramagnetic species come in a wide
range such as bio-macromolecules like DNA31 ,proteins32 and recently lipids33; the GdDOTA(GAC12)2
complex displayed the highest relaxivity, r1, reported to date for a Gd-based paramagnetic micelles
[34.8 mM-1s-1at 25°C and 20 MHz (0.5 T)]. There are also those based around a synthetic nature such
as polymers34 and dendrimers35.
One suggested method of increasing τr is to position the Gd3+ ions within the barycentre of
the macromolecule. This transfers the molecular tumbling of the molecule into a rotation about the
Gd-H2O bond vector. An example of this can be seen in the modified [Gd(DOTA)(H2O)]- complexes
shown in Figure 14. [Gd(DOTA)(H2O)]- which has a relaxivity value of ~4mM-1s-1 (25°C, 20 MHz),
whereas the modified [Gd DOTA-Glu12 (H2O)]5- complex has a relaxivity of ~23.5 mM-1s-1 (25°C, 20
MHz). This high relaxivity value is due to obtaining an optimal τr of the complex through the increase
in molecular weight by a factor of five.27, 36
24
Figure 14. [Gd DOTA-Glu12 (H2O)]5- (left) and [Gd DOTA-Glu12Gly4(H2O)]5- (right) have optimal τr values, due to the location of the Gd3+ ion on the barycentre of their macromolecular structure.
The contribution from the second and outer sphere parameters can also lead to an increase
in relaxivity of up to 20% in some Gd3+ complexes.37, 38 It has been estimated that the residence time
of the second sphere water molecules, τm ss, is in the picosecond range, which is considerably short
than the residence time of the directly coordinated water molecule, τm. Another physical parameter
stemming from SBM theory that also contributes towards the overall relaxivity is The Gd-H distance
which also have a significant effect on the inner sphere proton relaxivity; it was found that a
decrease of 0.1Å in the Gd-H distance corresponded to a 20% increase in the inner sphere relaxivity,
whilst a decrease of 0.2Å resulted in as much as a 50% increase.39
Contrast agents can be further enhanced by creating pH, metal ion, enzyme or biomolecule
sensitive contrast agents. For contrast agents to be effective within the human body it must be non-
ionic to minimize osmolality, but have good water solubility for efficient transport around the body,
a low toxicity whilst retaining a high relaxivity, and possess thermodynamic, kinetic and in vivo
stability.
25
36
Typical T1 contrast agents used in MRI scans have; one coordinated water molecule, a τm
value over 100ns and a τr value of 0.1ns, which results in them having low relaxivities of about 4-5
mM-1s-1.10,27,40 These relaxivity values are up to twenty times lower than the theoretical values
obtainabed by optimising the parameters according to SBM theory. Some Gd3+ contrast agents that
have been reported to have improved relaxivities as a direct result of the optimisation of chemically
controllable parameters from SBM theory include; a Gd(III) TREN-Me-3,2-HOPO complex with a
relaxivity value of 10.5 mM-1s-1; 41 a texaphyrin Gd3+ complex with a relaxivity of 16.9–19 mM-1s-1;42 a
β-cyclodextrin “click cluster” containing seven paramagnetic Gd3+ chelates with a high relaxivity of
43.4 mM-1s-1 per molecule and 6.2 mM-1s-1 per Gd3+ at 9.4T;43 a tetranuclear Gd3+ complex of DO3A
(1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide) attached to a pentaerythrityl
framework with a high relaxivity, r1 of 28.13 mM-1s-1 (24 MHz, 35°C, pH 5.6), it also has a transverse
relaxivity, r2, of 129.97 mM-1s-1 (24 MHz, 35°C, pH 5.6) which means it could have potential use as a
T2-weighted contrast agent. 44 These are prime examples that show through the optimization of the
three main parameters; the residence time of the coordinated water molecule, the rotational
correlation time and the hydration number, a huge increase in the relaxivity can be obtained.
26
1.6 Contrast Agents
The contrast in the MRI image is due to the differences in relaxation rates of water protons
in tissues. MRI was originally expected to be a medical imaging technique that could make
conclusive diagnoses whilst being non-invasive, however the contrast produced was very poor, this
was the case up until it was found that contrast agents could significantly enhance the contrast
quality. Contrast agents can be administered orally or more commonly through intravenous
injection, which enhances the appearance of soft tissues such as the brain, the heart, muscles, blood
vessels and even tumours. Tumours show up on an MRI because the contrast agent enhances the
distinction between normal and abnormal tissue.45 The images acquired from MRI can lead to the
diagnosis of many diseases early on in their development, which is why it is such an important
technique used in medicine. Blood flow through certain organs and blood vessels can also be
monitored using specific contrast agents allowing problems with blood circulation, such as blockages
or a rupture in the blood vessel, to be identified.46
Contrast agents are mainly paramagnetic, but some have ferromagnetic or
superparamagnetic properties. Ferromagnetic and superparamagnetic contrast agents both contain
nanoparticle clusters of iron, which is normally bound to an organic substrate, which can produce
magnetic moments up to 1000 times larger than that of protons.47Both ferromagnetic and
superparamagnetic complexes alter the contrast of an image by producing inhomogeneities in the
magnetic field, Bo, around the ferromagnetic medium; this in turn reduces the T2 relaxation times of
the water molecules around the contrast agent48 therefore giving a negative contrast.49
Ferromagnetic species Maintain their magnetic moment even after the external magnetic field is
taken away but paramagnetic and superparamagnetic lose their magnetic moment once the external
magnetic field is removed. Typically, particles displaying superparamagnetism rather than
ferromagnetism are used, as these display better contrasts due to the increase in relaxivity.
Due the combination of all the individual domains of the unaligned magnetic moments
within a Paramagnetic metal such as gadolinium, iron, chromium and manganese, they all have
permanent magnetic fields.50 when an external magnetic field is applied to a paramagnetic species,
the domains of the unaligned magnetic moments become aligned and this generates a strong local
field which can be up to 104 gauss.51 Paramagnetic contrast agents decrease the T1 and T2 relaxation
times of water molecules that they interact with by generating time varying magnetic fields; This
decrease in relaxation times occurs due to the tumbling of the water-metal complex and the
electron spin flips of the unpaired electrons in the paramagnetic metal.
27
1.6.1 Gadolinium
[Gd(DTPA)H2O]2- (r1 = 4.5 mM-1s-1at 20MHz and 37°C)52 was first defined in 1971 and in 1988
it was approved as an MRI contrast agent. Since then contrast agents based on Gadolinium are by far
the most used and widely researched today. The reason for this interest in Gd3+ for use as a metal
centre in a contrast agent is due to certain properties is possesses; firstly it is the only ion with 7
unpaired electrons, which gives it a great magnetic moment. although the magnetic moment has an
effect on the relaxation rate of Gd3+, it is the highly symmetrical configuration of these electrons in
its electronic ground state that gives the ion its long electron spin relaxation time.25 dysprosium3+ for
example has a much larger magnetic moment than Gd3+ but the electronic ground state of Dy3+ ion is
asymmetric and causes it to exhibit a short electron spin relaxation time. For these reasons
gadolinium is the most used metal for MRI and it can be considered the most effective metal in the
enhancement of T1 relaxation rates of water protons.10, 27, 53
Although these properties make it ideal for use in MRI , Gd3+ as a free ion is very labile and
highly toxic within the body. For these reasons it must be administered as a very stable chelated
complex so that the metal ion will not be released in vivo. Gd3+ has a coordination number of nine
and current medically approved Gd3+ contrast agents are bound to an eight coordination ligand such
as DTPA (diethylenetriaminepentaacetic acid) with a formation constant of ~22-23,52 or DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Figure 15). EDTA is a six coordinate
ligand (ethylenediaminetetraacetic acid)but it is also known to form stable complexes with Gd3+ and
has a reasonably strong formation constant (log k) of ~17.52 The remaining coordination sites on the
metal centre are taken up by water molecules.
Figure 15. Ligands that form stable chelated complexes with Gd3+
28
EDTA DTPA
DOTA DO3A
In 2006 a connection was determined between the use of Gd3+ contrast agents and
nephrogenic systemic fibrosis (NSF).54,55 NSF is a rare systemic fibrosing disorder which can affect the
skin, joints, eyes, and internal organs. The first case ever identified was in 1997 and its cause is not
fully understood. However there is evidence to suggest that this disorder is associated with a
prolonged exposure to gadolinium in patients with severe kidney failure.56 Then in 2007 the
European Medicines Agency classified contrast agents containing gadolinium into three categories
based on their structures; those which are least likely to release free Gd3+ ions in the body have a
cyclical structure those which are ‘somewhat likely’ to release free Gd3+ ions in the body have an
ionic linear structure and those which are “most likely” to release free Gd3+ ions in the body have a
linear non-ionic structure. Finally in 2009 Broome. summarized the medical literature reporting of
biopsy-proven NSF cases in which the authors specifically investigated patient exposure to Gd-CA.57
1.6.2 Manganese
Manganese ions also contain unpaired electrons giving them paramagnetic properties which
enhance T1 relaxation via interaction with water protons local to them. Manganese ions have several
other properties that make them suitable to be used as an MRI contrast agent; a high spin number, a
long electronic relaxation rate and a labile water exchange.58 The most efficient manganese ion is the
Mn2+ as it has five unpaired electrons in the 3d orbital and like Gd3+ it also has a symmetrical
electronic ground state giving it a slow electron spin relaxation. Mn2+ complexes are known to be
less thermodynamically stable than Gd3+ complexes due to the Mn2+ high-spin d5 electron
configuration having no ligand field stabilisation. Even though the complexes are less
thermodynamically stable than Gd3+, Mn2+ is an essential trace element existent in all human cells
and so it is less toxic and it is assumed that less attention needs be given to the release of
manganese ions in the body. Mn3+ has also been used to form the porphyrin complex [Mn-TPPS4].50
There are, however, concerns about the stability of manganese complexes when used in high doses
for MRI, this is because Mn3+ can bind to transferrin and cross the blood brain barrier by transport
mechanisms.59Also Mn2+ can bind to α-macroglobulin and which is known to collect in the brain60 and
can lead to neural syndromes such as Parkinson’s disease.61
29
Commercially used Mn2+ contrast agents include the gastrointestinal imaging agent
Lumenhance62,63 (although it has now been discontinued), MnCl2 (r1 = 8.0 ± 0.1 mM-1s-1 at 20MHz and
37°C)64 However the only Mn2+ contrast agent still commercially available today and the only one to
have been approved to be administered via intravenous injection is the liver imaging agent Teslascan
[Mn(DPDP)]4- (Figure 16).65,66 [Mn(DPDP)]4- has no water molecules coordinated to it and so the
overall relaxivity could actually be caused by Mn2+ being released into the body as a free ion. The
role of the (DPDP)6- ligand is to delay the release of the metal ion into the human body and hence
decrease toxicity, however at the same time it could help enhance the relaxivity by increasing the
rotational correlation time.
Figure 16. [Mn(DPDP)]4- (DPDP)6-= N,N’-dipyridoxylethylenediamine-N,N’-diacetate- 5,5’-
bisphosphate)
1.6.3 Iron
The transition metal, iron, also contains unpaired electrons but iron as an oxide belongs to a
group of superparamagnetic materials which affect T2 relaxivity. Even though Fe3+ has five unpaired
electrons, it is not directly comparable to Mn2+ because iron oxide is administered as slightly soluble
particles instead of a water soluble complex. Nano particles of iron oxide display superparamagnetic
properties whilst the larger particles of iron oxide are ferromagnetic. These nano particles are
commonly known as superparamagnetic Iron Oxide particles or SPION’s.67 Iron is therefore of some
suitability for use in MRI.
30
1.6.4 Macromolecular contrast agents
It has been well established that for the majority of linear polymeric contrast agents with
molecular weights of the contrast agent to be in excess of 10,000 g mol-1, only a small increase in the
rotational correlation time is observed. This is because at these high molecular weights there is
flexibility within segments of the polymer and this governs the tumbling motion of the paramagnetic
species.68 An increase in any internal flexibility also results in a decrease in the overall relaxivity of
the complex.10 Duarte et al. Also found that conformationally more rigid and cyclic polymeric
contrast agents had a higher overall relaxivity compared to linear and conformationally flexible
polymeric contrast agents.69
The most common and iconic methods for making high relaxivity contrast agents is to
conjugate functionalised low molecular weight complexes such as, [Gd-DTPA] or [Gd-DOTA], to
macromolecules. This changes the physico-chemical parameters associated with the low molecular
weight complexes; the main parameter that is affected is the rotational correlation time.70 General
conjugation methods include functionalization of primary amines via alkylation, reductive amination,
acylation and thiourea formation.
Now synthetically made linear polymers, such as polylysine (PL), are available in many
molecular weight groups. The amino groups on the lysine backbone can be modified with
acyclic/macrocyclic polyaminocarboxylate derivatives such as DTPA or DTTA-MA (diethylenetriamine
tetraacetic acid monoamide). [Gd-DTPA-PL] as synthesised by Schuhmann et al. has a relaxivity range
of 15-20 mM-1s-1 depending on its molecular weight (Appendix 10).71 This relaxivity is up to four
times higher than that of [Gd-DTPA].There are two types of PL contrast agents; low molecular weight
which have rapid excretion rates and high molecular weight which have much longer blood pool
half-lives.72 The disadvantage of the DTPA-modified PL contrast agents arise from the fact that they
have a longer circulation time in the kidneys when it comes to excretion. Polylysine has also been
copolymerised with PEG (polyethylene glycol) and the relaxivity of these complexes is shown also
shown in Appendix 10.
Even more Recently gadolinium complexes have been grafted onto virus capsids.73,74,75 One
of the highest relaxivities ever recorded for a virus capsid agent, is that of a the (TREN(HOPO)2(TAM)
chelate attached onto bacteriophage MS2 capsids.76 It has 90 Gd3+ complexes per capsid and a
relaxivity per gadolinium of 30.7 - 41.6 mM-1s-1 (30MHz, 25°C). The relaxivity per particle is 2500 -
3900 mM-1s-1 (30MHz, 25°C). The relaxivity is based on the rigidity of the complex and generally the
more rigid the complex the higher the relaxivity.
31
The protein, human serum albumin, (HSA), has also been used to non-covalently bind low
molecular weight complexes in vivo.77 The blood residency time and rotational correlation time was
increased when the low molecular weight contrast agent was bound to HSA. This lead to a superior
contrast enhancement for magnetic resonance angiography (MRA). The high relaxivity (20-45 mM-1s-
1, 20MHz) displayed by [Diphenylcyclohexyl phosphodiester-Gd-DTPA] (Trade names; Vasovist or
Ablavar) is due to non-covalent bonding to HSA in vivo.7879,80,88 When a PEG group (chain length = 122)
was attached to Vasovist, the relaxivity increases to 74±14 mM-1s-1 at 20MHz.81 This is due to an
increased rotational correlation time associated with the increase in molecular weight.
1.7 Chitosan
Chitosan (Figure 17) is a linear polysaccharide made up of β-(1-4)-linked D-glucosamine and
N-acetylglucosamine units which are randomly dispersed. It is a derivative of one of the most
abundant natural biopolymers in the world, chitin (Figure 18). Chitin is a polymer of N-
acetylglucosamine and can be found in the shells of crustaceans such as crabs, shrimp, the cell walls
of fungi and even the exoskeleton of insects. Chitin is structurally very similar to cellulose; the only
difference is that it has one hydroxyl group substituted for an acetyl amine group on each monomer.
This results in an increased strength of the chitin-polymer matrix through increased hydrogen
bonding between neighbouring polymers chains. Chitosan is produced commercially through the
deacetylation of chitin and can be anywhere between 60-100%. The molecular weight of
commercially produced chitosan can be anywhere between 50,000 and 375,000g.
Chitosan has been a polymer of interest due to its non-toxic, biodegradable and
biocompatible properties. It has also been found to have antifungal82, antimicrobial83 and antitumor84
properties which make it an ideal candidate for use in medicine. Chitosan is also used in a wide
range of other applications. When being used to remove heavy metals in waste water chitosan act as
a chelating agent due to the presence of the amino groups on the polymer chain. Copper, Mercury,
Zinc, Cadmium and Nickel have been found to have a strong affinity with chitosan.85 The binding
strength of the metal ions to chitosan can be affected by many factors, such as the pH of the
solution, the degree of deacetylation or the form of the chitosan polymer e.g. film, powder, fibres,
flakes.86
Chitosan is insoluble in water and most organic solvents. However it can be made soluble in
acidic solutions, where the pH is lower than 6 and the degree of deacetylation is over 50%.87The
reason for this solubility in these conditions is due to the protonation of the amino groups on the
32
chitosan backbone, which has a pKa value of ~6.5, when in solution the polymer is transformed into
a polyelectrolyte.
The degree of deacetylation is the percentage of acetyl groups present on the polymer chain
and can be calculated using equation (17). Even though the polymer is soluble in the conditions
mentioned above, it has the tendency to precipitate out of solution due to the acetyl and hydroxyl
groups deionising and becoming hydrophobic.86, 88
% degreeof deacetylation=N (amine group)
N (monomer of Chitosan)×100(17)
The degree of deacetylation is a vital characteristic of chitosan that affects both its chemical
and physical properties87 and it can be determined by NMR and IR spectroscopy.89A common method
of deacetylation uses sodium hydroxide in excess as a reagent and water as a solvent. When allowed
to go to 100% deacetylation this reaction method can yield up to 98% product.90 The degree of
deacetylation is an important factor when calculating the molecular weight of the polymer, as it has
a strong impact on the physicochemical, biological and rheological properties of the polymer.91
Methods of molecular weight determination include gel permeation chromatography92 and light
scattering93
Figure 17. Chitosan structure
Figuge 18. Chitin structure
33
2. Experimental
2.1 Materials
All reagents and solvents were commercially available and were used without further purification.
Chitosan (medium molecular weight, 190,000 – 310,000 g mol-1, 75-85% deacetylated),
diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), gadolinium
chloride hexahydrate (GdCl3.6H2O), sodium hydroxide, deuterated water, hydrochloric acid and its
deuterated form (DCl) were acquired from Aldrich. The complexes of [Ch-DTPA] and [Ch-EDTA] were
prepared earlier in the year by the previous undergraduate and these samples were used to keep
the results comparable with previous data. The complexes were analysed by 1H NMR and FTIR to
ensure that these complexes hadn’t degraded over time. The preparation and reaction schemes
used are shown by Appendix 7, Appendix 8 and Appendix 9. All other chemicals and materials were
readily available from the Inorganic Chemistry and the Solid State and Materials Chemistry
Laboratories.
2.2 Instrumentation and Methods
All syntheses of the modified chitosan polymer, Gd3+ and Mn2+ complexes were performed in
air. All relaxivity measurements were recorded on a STELAR Spinmaster FFC-2000 relaxometer. All 1H
NMR spectroscopy measurements were performed on a Bruker 500 MHz NMR spectrometer using
1% (w/w) solutions of Chitosan, Chitosan-DTPA and Chitosan-EDTA, which were prepared in 2mL of
deuterated water with the pH adjusted to 4.7 using DCl. All IR spectra were recorded on SHIMADZU
IR affinity-1,fourier transform infrared spectrophotometer and the samples were analysed solvent
free.
Small Angle Neutron Scattering (SANS) data were obtained from the SANS 2D instrument at
the ISIS facility, Oxford, UK, using 1% (w/v) solutions of chitosan, Chitosan-DTPA and Chitosan-EDTA,
which were prepared using the same method as described for NMR measurements. These
experiments were carried out by A. Paul and the data provided was in the standard reduced format
of Q and I(Q) for interpretation. Error bars were not included in the relaxivity data and SANS data as
the errors associated with these spectroscopic techniques are so small relative to each data point
that they have no significance. Also when these errors were included in the graph it obscured the
data points.
34
2.3 Preperation of the control groups
2.3.1 Preparation of Chitosan and Gadolinium as a physical mixture
A solution of chitosan (80.6 mg, 5mM) was made in deionised water (100mL) (with 1% v/v of
HCl 6M) and the pH was set to 4.7 using NaOH (1M) when the chitosan had dissolved. Known masses
of GdCl3.6H2O were then added separately to known volumes of chitosan solution (5mM) to give a
range of Gadolinium concentrations from 0.5mM-25mM.
2.3.2 Preparation of Chitosan and Manganese as a physical mixture
A solution of chitosan (80.6 mg, 5mM) was made in deionised water (100mL) (with 1% v/v of
HCl 6M) and the pH was set to 4.7 using NaOH (1M) when the chitosan had dissolved. Known masses
of MnCl2.4H2O were then added separately to known volumes of chitosan solution (5mM) to give a
range of Manganese concentrations from 0.5mM-25mM.
35
2.4 Relaxivity measurements
The proton relaxivities, r1, of the polymeric contrast agent [Ch-DTPA-Mn], and both physical
mixtures of [Ch-Mn] and [Ch-Gd] were obtained at 25°C and 37°C using FFC relaxometry at various
field strengths between 10 KHz and 30 MHz on a STELAR Spinmaster FFC-2000 relaxometer.
2.4.1 Relaxivity measurements of Mn2+ bound to the modified Chitosan [Ch-DTPA-Mn]
A solution of MnCl2.4H2O (10mL, 5mM) containing DTPA-Ch (1mM with respect to the
assumed DTPA-Ch monomer shown in Scheme 2) and a solution of just DTPA-Ch (10mL, 1mM with
respect to the assumed DTPA-Ch monomer shown in Scheme 2) were made up. Known aliquots of
the Manganese containing solution were titrated into the solution of DTPA-Ch and the relaxivity
measurements of the new solution were recorded after each addition. This was repeated until an
excess amount of Gd3+ had been added.
2.4.2 Relaxivity measurements of Gd3+ and Chitosan as a physical mixture
Relaxivity measurements were recorded for Chitosan solutions (5mM) containing differing
Concentrations of Gadolinium ions, 0.5mM-25mM.
2.4.3 Relaxivity measurements of Gd3+ and Chitosan as a physical mixture
Relaxivity measurements were recorded for Chitosan solutions (5mM) containing differing
Concentrations of Manganese ions, 0.5mM-25mM.
36
3. Results and discussion
The potential MRI contrast agent [Ch-DTPA-Mn] was synthesized through a cross coupling
reaction between the chelating ligand, DTPA and the polymer, Chitosan. This was then followed by
coordination of the [Ch-DTPA] complex with Gd3+ and Mn2+ ions. Darras et al104 had previously
synthesised and studied the complex [Ch-DTPA-Gd] but they did not obtain any relaxivity data of the
complexes. The synthesis was repeated using a slightly modified method to include these relaxivity
measurements.
3.1 Synthesis of the modified chitosan polymers
The modified chitosan polymers, [Ch-DTPA] and [Ch-EDTA], were synthesised by a previous
undergraduate, Bhanu Partap Singh Rana. This [Ch-DTPA] complex that he synthesised was used for
the synthesis of the final polymeric contrast agent. The reaction schemes and preparations of the
modified chitosan polymers that he used are shown in appendix7, 8 and 9. These samples were
analysed by IR and 1H NMR spectroscopy to verify that they had not degraded over time and were
suitable for use in the synthesis of the polymeric contrast agent.
Rana found that after the synthesis of the [Ch-DTPA-Mn] solution the complex had a
tendency to precipitate out of solution1, this could be down to the fact that the pH of the solution
was set to 4.7 which would decrease the solubility of the polymer and cause it to precipitate out of
solution. This synthesis was repeated but once the modified chitosan polymer had been dissolved
into solution the pH was kept slightly more acidic than the pH 4.7 that was specified by Rana, which
increased the solubility of the polymer as it more readily formed the chitosan polyelectrolyte.
37
3.2 FT-IR data of Chitosan, Ch-DTPA and Ch-EDTA
During the cross coupling reaction an amide bond is formed between a carboxylic acid group
on the Chelating ligand and an amine group on Chitosan, which show characteristic absorptions on
an IR spectrum. These characteristic peaks are attributed to an amide I and II band observed at
around 1650 and 1550 cm-1 respectively. So the presence of the amide I and II band can prove that
the DTPA chelating ligand is covalently bonded to the chitosan polymer via an amide bond. This
amide II band has been observed in a previously synthesised complex of Gd-DTPA complexed to
chitosan by Huang et al.94 However 75-85% deacetylated Chitosan also contains amide bonds from
the acetyl groups, so an observed amide I and II shoulder peak will be seen in all the complexes that
were analysed, therefore if these peaks are present it will only give an indication that an amide bond
has formed between Chitosan and DTPA.
The IR spectra of Chitosan with peak assignments are shown in Appendix 1 and its values are
in agreement with values stated in literature.94, 95, 96 Darras et al. have previously characterised
Chitosan and DTPA complexes by IR spectroscopy, figure 19, they found that in the DTPA ligand the
bands that are observed at 1730, 1696 and 1630cm-1, correspond to (C=O) vibrations of the COOH
group in the monomer, dimer and and carboxylate form, respectively.95
The IR spectra of the synthesised Ch-DTPA (Appendix 3) is very similar to that of 75-85%
deacetylated chitosan, with bands observed at 1655cm-1 corresponding to (C=O) and (C–O)
vibrations and bands between 1034 and 1074cm-1 corresponding to (C–O–C) vibration on the
chitosan backbone. The amide I and II shoulder peaks for all spectra cannot be easily seen due to the
complexity of the peaks around that band area, so conformation that the amide bond had formed
could not be fully justified. However there is a slight shift of peaks around the amide I and II band
area when comparing the IR of Chitosan and Ch-DTPA, this is a good indication there has been a
slight conformational and structural change of Chitosan after it was reacted with DTPA and that an
amide bond has formed as suggested by Chung et al.97 EDTA is structurally similar to DTPA but is
essentially a smaller version of the complex so the same bands are seen in the IR spectra of Ch-EDTA
(Appendix 5); with the (C=O) and (C–O) band at 1630cm-1 and at around 1560cm-1.
38
Figure 19. FT-IR spectra of 96% deacetylated chitosan, DTPA, a physical mixture of
chitosan and DTPA and a modified chitosan with 20% (mol/mol) DTPA.
3.3 1H NMR data of Chitosan, Ch-DTPA and Ch-EDTA
All three samples were characterised by 1H NMR spectroscopy and these are shown in
Appendix 2, 4 and 6. The chemical shifts obtained for these complexes are in agreement with
literature values.104 Due to the similarities in the structure of these complexes the NMR spectrums
had similar peaks. The signal at ~ 4.6 ppm was assigned to the resonance of the anomeric protons on
Carbon 1 of the D-glucosamine units (deacetylated unit), the signal at ~ 2.00 ppm to the three
protons of the N-acetyl-D-glucosamine units (acetylated unit) and the signals at 3.50 - 4.00 ppm to
the rest of the protons of the D-Glucosamine units of the Chitosan polymer. Signals at ~ 3.75, 3.25
and 3.2 ppm were attributed to protons D, C and A of the DTPA and EDTA ligand, respectively. The
signal between ~ 2.70 and 3.10 ppm was due to an overlap of the chitosan protons on Carbon 2 and
B protons of DTPA and EDTA. There is a difference in the chemical shift of 0.15 ppm of the proton on
carbon 2 between pure Chitosan and the modified Chitosan complexes which suggests that there
was a change of chemical environment of the proton on this carbon , so there is good assumption
that this change was due to the covalent amide bond formed between Chitosan and the chelating
ligands.95 However due to the low concentrations of the samples and fairly low loading values
obtained from the relaxivity data, quantitative analysis to determine the loadings of the chelating
agents bound to chitosan was not able to be performed using the NMR data.
39
3.4 Relaxivity Data
For a paramagnetic contrast agent there is a linear proportionality between the solvent
longitudinal relaxation rate (1/T1) and the paramagnetic species concentration [M]. This relationship
is described in equation (18).
(1/T1)obsd = (1/T1)d + r1 x [M] (18)
Where (1/T1)obs is the measured solvent relaxation rate in the presence of the paramagnetic
species, (1/T1)d is the measured solvent relaxation rate in the absence of the paramagnetic species,
[M] is the concentration of the paramagnetic species and r1 is the relaxivity. This relaxivity has units
of mM-1s-1 and is defined as the gradient of (1/T1)obsd against [M].98
Relaxivity, r1, is defined as the change in relaxation rate of the solvent water protons upon
addition of a contrast agent and is a result of the alteration of the dipolar interaction between the
magnetic moment of the metals unpaired electrons and water protons. Certain parameters
stemming from SBM theory can be altered to enhance this relaxivity, which was explained in detail
earlier in the report. The main parameters that can be controlled are; the residence time of the
coordinated water molecule (τm), the rotational correlation time (τr), the electronic relaxation of the
paramagnetic species (T1e/T2e), the hydration number (q), and the distance between the coordinated
water molecules and the paramagnetic species.
It was predicted that the contrast agent synthesised would have an increased rotational
correlation time due to the incorporation of a high molecular weight polymer; so this can be
considered as the most important parameter for the increase in relaxivity of that specific contrast
agent. However it was also predicted that the contrast agent has no water molecules directly
coordinated to the paramagnetic species, which results in a hydration number of zero and hence
there will be no residence time associated with the coordinated water molecule. So it can be
considered that these parameters have no role in the enhancement of the relaxivity of the specific
contrast agent. The hydration number and residence time parameters have a greater effect in the
enhancement of relaxivity than the rotational correlation time and so it can also be predicted that
the contrast agent [Ch-DTPA-Mn] would have the lowest relaxivity value when compared to the
relaxivities of [Ch-DTPA-Gd], [Ch-EDTA-Gd] and [Ch-EDTA-Mn]. This prediction is based on the fact
that all the other complexes have at least 2 water molecules directly coordinated to the
paramagnetic species as well as the increase in the rotational correlation time due to the polymer.
40
It was assumed by Rana that all Gd3+ and Mn2+ ions bound to DTPA-Ch and EDTA-Ch is
through chelation with DTPA and EDTA.1 So relaxivity data for Chitosan in the presence of just the
Gadolinium and Manganese ions separately were recorded; these were assigned as control groups
for the experiment. This was done to verify the assumption he made by determining whether or not
the paramagnetic species was coordinating to the chitosan backbone as well as the chelating ligands.
This determination comes about due to the differences in relaxivities between free and bound form
of the paramagnetic species; the free form will always possess a steeper slope and hence a larger
relaxivity than the bound form. If Chitosan was not coordinating to the paramagnetic species a
linear relationship would be observed in the relaxivity data.
It is also worthy to note that the loading values of the paramagnetic species onto the
Chelating ligands can be calculated from this relaxivity data and hence the loading values of these
ligands onto Chitosan can be obtained; on a relaxivity graph this is the concentration of the
paramagnetic species at the point of intersection between the two slopes. If the assumption made
by Banhu was incorrect and there was coordination between the paramagnetic species and Chitosan
then the total loading values of the paramagnetic species that was calculated from both colorimetric
analysis and relaxivity data would be the sum of the loading values onto Chitosan and DTPA, so this
is not a true representation of the loading values onto just the ligand, hence the optimal loading
percentages of the Paramagnetic species onto the ligands would be incorrect and so optimal
relaxivities would not be achieved. However Banhu found that these colorimetric assays gave
random and inconclusive results which were assumed to be caused by the arsenazo I dye interacting
with the modified polymer somehow. For this reason colorimetric analysis was not used or reported
as a technique for the calculation of loading percentages.
The assumption made is in accordance with Darras et al. Where they stated that Chitosan
does not coordinate with Gd3+ at all.95The control groups were set up to verify this statement so that
the optimal loading percentages of the ion onto the ligand could be correctly calculated from the
relaxivity data. It is assumed that the chelating ligands are attached by only one amide bond to the
chitosan backbone as shown in Scheme 1 and 2. But according to Caravan et al. the complexation
capabilities of the chelating ligands, DTPA and EDTA largely depend on the geometry that the
molecules are in.99 From this, it can be determined that DTPA and EDTA, covalently bonded to
chitosan, can still complex Gd3+ ions at the percentages found by Rana.1
41
The spin-lattice relaxation was measured under the same conditions (10 KHz – 30 MHz, 25°C)
for the Control groups and the [Ch-DTPA-Mn] complex. The r1 values were obtained and these were
used to compare [Ch-DTPA-Mn] to the other previously made contrast agents [Ch-DTPA-Gd], [Ch-
EDTA-Gd], [Ch-EDTA-Mn] and to that of the first FDA approved contrast agent, [Gd-DTPA]
(Magnevist, 3.4 – 4.5mM-1s-1, 1.5T, 25°C). Magnevist is commonly used as a reference in the
development of novel contrast agents.25 for comparative reasons [Gd-DTPA] has a low rotational
correlation time due to its low molecular weight but it has one water molecule coordinated to the
paramagnetic species in the inner sphere. Finally the ligands DTPA and EDTA are known to form
stable complexes with gadolinium; the formation constants (log k) for these complexes are 17 and
22.5 respectively.52
3.4.1 Relaxivity Data of the Control groups
The relaxivity data for the control groups was carried out using Chitosan (5mM) over a range
of Gd3+ and Mn2+ concentrations (0.05, 0.10, 0.25, 5.00, 10.00 and 25.00 mM) these high
concentrations were used to aid in the production of the Chitosan-Gd complex by forcing the
equilibrium over to the product side. If the relaxivity data of these physical mixtures shows that no
paramagnetic has coordinated to the Chitosan at these high concentrations this would suggest good
evidence that there will be no complexation between the two molecules at any given concentration.
The relaxivity measurements for [Ch-Gd3+] (physical mixture) are shown in Figure 20.
Figure 20. Relaxivity measurements of Gd3+ and Chitosan as a physical mixture.
Figure 20 shows the relaxivity of [Gd3+-Ch] to be 11.18 mM-1s-1 at 30 MHz and 25°C as
calculated by the gradient. This relaxivity value is much greater than that of Magnevist, which can be
attributed to free Gd3+. There is a linear relationship between the 1/relaxation time and the
concentration of Gd3+ which is definitive proof that Gd3+ does not complex to Chitosan; if Chitosan
was complexing with the paramagnetic species then there would be a change in the gradient and
hence the relaxivity due to the differences in relaxivity of bound and free paramagnetic species.
Relaxivity data at 37°C and 10kHz was also recorded for this sample and a linear relationship was
observed in all cases.
The relaxivity measurements for [Ch-Gd3+] (physical mixture) are shown in Figure 21.
42
0.00 5.00 10.00 15.00 20.00 25.00 30.000.0000
20.0000
40.0000
60.0000
80.0000
100.0000
120.0000
140.0000
160.0000
180.0000
f(x) = 6.7502336301122 x + 0.22169364458923R² = 0.998632370488528
relaxivity of Mn2+-Chitosan (physical mixture)(carried out at 25°C, 30MHz)
concentration of Mn2+ (mM)
1/ T
1 (s
-1)
Figure 21. Relaxivity measurements of Mn2+ and Chitosan as a physical mixture.
Figure 21 shows, the relaxivity of [Mn2+-Ch] to be 6.75 mM-1s-1 at 30 MHz and 25°C as
calculated by the gradient. This relaxivity value is greater than that of Magnevist, which can be
attributed to free Mn2+. There is a linear relationship between the 1/T1 and the concentration of
Mn2+ which is definitive proof that Mn2+ does not complex to Chitosan. Relaxivity data at 37°C and
10kHz was also recorded for this sample and a linear relationship was observed in all cases.
3.4.2 Relaxivity Data of [Ch-DTPA-Mn]
The relaxivity measurements for [Ch-DTPA-Mn] are shown in Figure 21. In [Ch-DTPA-Mn] it is
assumed that there are no water molecules coordinated to the paramagnetic Mn2+ ion. This comes
from the fact that Mn2+ has seven available coordination sites, all seven of which are taken up by the
chelating ligand bound to chitosan, leaving the no sites on the paramagnetic species free to bind a
water molecules.112
43
0.18 0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.360
0.5
1
1.5
2
2.5
3
f(x) = 8.55392758197326 x − 0.405713238720314R² = 0.996843375736677
f(x) = NaN x + NaNR² = 0 Relxivity of [Ch-DTPA-Mn]
(carried out at 25°C, 30MHz)
Bound MnLinear (Bound Mn)Free MnLinear (Free Mn)
Conc. of Mn2+ (mM)
1/T1
(s-1
)
Figure 22. Relaxivity measurements of [Ch-DTPA-Mn] where the blue line represents Mn2+
bound to Ch-DTPA and the red line represents free Mn2+ ions in solution.
Figure 22 shows, the relaxivity of [Ch-DTPA-Mn] to be 3.85 mM-1s-1 at 30 MHz and 25°C as
calculated by the gradient. This is lower than that of Magnevist and this decrease in relaxivity can be
directly associated to the absence of water molecules directly coordinated to the paramagnetic
species in the inner sphere. This lowers the hydration number (q) and residence time (τm)
parameters which have the biggest effect on the overall relaxivity of a contrast agent. However
there is an increase in the rotational correlation time due to the incorporation of a high molecular
weight polymer, which is why this complex displays a relaxivity close to that of Magnevist.
It can also be observed that from the point of intersection, 1mM Ch-DTPA is fully saturated
and has bound ~ 0.18 mM Mn2+. The relaxivity data on the control groups show that there is no
complexation of the paramagnetic species to chitosan and assuming a 1:1 ratio of metal to chelating
ligand, this corresponds to an 18% loading of DTPA on chitosan.
There is an interesting property associated with manganese ions, if we look at the relaxivity
measurements of [Ch-DTPA-Mn] at a low field (10 KHz) as shown in Figure 23, we can see that the
relaxivity is vastly improved.
44
Figure 23. Relaxivity measurements of [Ch-DTPA-Mn] where the blue line represents Mn2+
bound to Ch-DTPA and the red line represents free Mn2+ ions in solution.
In Figure 22 at a low field of 10 KHz it can clearly be seen that the relaxivity measurements
of [Ch-DTPA-Mn] display a greater relaxivity value of 20.08 mM-1s-1 than when at 30MHz. This is a
unique property associated with the paramagnetic Mn2+ ion and the exact reasons for this high
relaxivity at low fields are still unknown. At low fields the point of intersection is very clearly defined,
therefore allowing easier and more accurate determination of the saturation values of the chelating
ligands. It can also be observed that from the point of intersection, 1mM Ch-DTPA is fully saturated
and has bound ~ 0.195 mM Mn2+. Again the relaxivity measurements carried out on the control
groups show that there is no complexation of the paramagnetic species to chitosan and there is the
assumption of a 1:1 ratio of metal to chelating ligand, so this corresponds to a 19.5% loading of DTPA
on chitosan.
3.5 Small angle neutron scattering Data (SANS) of Chitosan, [Ch-DTPA] and [Ch-EDTA]
The SANS measurements for Chitosan, [Ch-DTPA] and [Ch-EDTA] are shown together on
graph 5. All the samples were made up of 1% (w/v) solutions in 2 mL D2O with the pH adjusted to 4.7
using DCl. It was not possible to study samples including the Gd metal as it strongly absorb neutrons
instead of scattering them and so the SANS experiment would be hindered to the extent that viable
45
0.18 0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.360
2
4
6
8
10
12
14
f(x) = 60.1005344057469 x − 8.554416380273R² = 0.996642260892858
f(x) = NaN x + NaNR² = 0 relaxivity of [Ch-DTPA-Mn]
(carried out at 25°C, 10kHz)
Bound MnLinear (Bound Mn)Free MnLinear (Free Mn)
Conc. of Mn2+ (mM)
1/T1
(s-1
)
data would not have been obtained, for this reason no polymeric contrast agents were sent for
SANS testing. Figure 24 shows the relationship between Q vs I(Q)
0.001 0.01 0.1 10.001
0.1
10
1000
Graph to show the relationship between Q and I (Q) of Chitosan, [Ch-DTPA] and [Ch-EDTA]
Ch-DTPA Ch-EDTA Chitosan
Q / Å-1
I(Q
) /cm
-1
Figure 24. SANS measurements of Chitosan, [Ch-DTPA] and [Ch-EDTA]
In Figure 24 the straight line between 0.1 and 1 is due to incoherent background of H nuclei
from the Chitosan complex.
An alternative presentation of this data for Chitosan, [Ch-DTPA] and [Ch-EDTA] is shown on
graph 6. All the samples were made up of 1% (w/v) solutions in 2 mL D2O with the pH adjusted to 4.7
using DCl. Figure 25 shows the relationship between Q vs I(Q).Q2
46
0 0.1 0.2 0.3 0.4 0.5 0.6-0.0005
0.0005
0.0015
0.0025
0.0035
0.0045
0.0055Graph to show the relationship between Q and I (Q).Q2 of Chitosan,
[Ch-DTPA] and [Ch-EDTA]
Ch-DTPA Ch-EDTAChitosanQ / Å-1
I (Q
).Q
2
Figure 25. SANS measurements of Chitosan, [Ch-DTPA] and [Ch-EDTA]
This ‘Kratky plot’ highlights the differences between the 3 datasets, and the slight
differences in slope observed in Figure 24. The similarity in the shape of the curves between the
samples indicates that there is no significant change in the shape (i.e. conformation) of the polymer
in solution; however the difference in gradients does indicate small changes in size may be present.
These small changes in size could be associated to the differences in Complex structure between the
3 samples. A detailed mathematical analysis of the SANS data was beyond the scope and time
available for this project.
4. Conclusions
Firstly I have successfully synthesized the novel polymeric contrast agent [Ch-DTPA-Mn]. The
relaxivity, r1, of this polymeric contrast agent has been measured and compared to the commercially
available and widely used contrast agent, [Gd-DTPA] (Magnevist). The relaxivity data gathered for
47
the synthesised contrast agent suggests that it would give lower tissue resolution in MRI than
Magnevist, but this was predicted earlier in the report due to the absence of water molecules
directly coordinated to the paramagnetic species in the inner sphere; a large contributing parameter
to the enhancement in relaxivity. The polymeric contrast agent would also have higher retention
times than that of Magnevist and other commercially available contrast agents due to the complex
possessing higher molecular weights associated with the polymer. This would give a larger time
frame in which MRI scans can be performed and could also qualify them for use as Magnetic
Resonance Angiography (MRA) contrast agents.
The complexes chitosan, [Ch-DTPA] and [Ch-EDTA] were also characterised via 1H NMR, IR
and SANS. The characterisation by NMR and IR verified that the complexes had been successfully
synthesised. The three complexes displayed very similar shaped curves in the SANS data which
indicates that there is no significant change in the conformation of the polymer in solution. However
the slight differences in gradient of the Kratky plot (Figure 24) indicate that the complexes are
slightly different in size and therefore structurally different.
The polymeric contrast agent synthesised has some great characteristic properties that
would make it an outstanding MRI contrast agent at low magnetic field strengths (10kHz); however
the toxicity, which would be due to the release of Paramagnetic species into the body and any other
Harmful effects of the complex, would need to be thoroughly evaluated before its release as a
commercially available MRI contrast agent.
Secondly two control groups were developed, consisting of physical mixtures of [Ch-Gd] and
[Ch-Mn] and these were also characterised via 1H NMR and IR. Relaxivity measurements of the two
samples have also been recorded that determined there was no coordination of Chitosan to both
Mn2+ and Gd3+ represented by the linear relationship of the relaxivity graphs. The fact that Chitosan
doesn’t complex with either ion is extremely favourable as this allows accurate evaluation of the
loadings of the paramagnetic species onto the ligands and hence accurately calculate the loadings of
these ligands onto the Chitosan backbone. It has also been found and should be noted that the use
of Mn2+ to accurately evaluate the loadings of paramagnetic species on contrast agents has great
potential at low fields (10 KHz).
There is huge potential for future work in this project. Firstly the r1 relaxivity of the
polymeric contrast agents can be increased by finding the optimal loading values of the chelating
agents, DTPA and EDTA, to chitosan and thus finding the optimal loading values of the paramagnetic
species to the polymeric contrast agent. It can be taken further through optimisation of length and
molecular weight of the chitosan backbone which would also increase the relaxivity by optimising
48
the rotational correlation time of the contrast agent. Relaxivity data could also be carried out on
different pH solutions of the contrast agent to see if pH has an effect on the structure and
conformation of the polymer in solution. Finally more data could be obtained from SANS
experiments and therefore a more detailed study of SANS could reveal more information about the
polymeric contrast agents conformation, size and shape in solution.
Beyond the potential for the increase of relaxivity lies the prospective thought of
synthesising a ‘smart’ contrast agent through the addition of targeting and biochemically specific
molecules (i.e. protein targeting within the human body)thus ensuring that the agent accumulates in
the exact position of interest where an MRI scan needs to take place. Furthermore, advances in
technology and instruments could give an insight into to how coordination geometry affects
relaxivity.
Appendix
Appendix 1
FT-IR spectra of pure medium molecular weight Chitosan (75%-85% deacetylated)
49
Wavenumber (cm-1) Peak interpretation87, 100
1018 ν-(C-O-C) Chitosan backbone1057 ν-(C-O-C) Chitosan backbone1150 ν-(C-O-C) Bridging oxygen1306 ν-(C-O-C)1375 δ-(CH2)1560 δ-(NH) Amide II1638 ν-(C=O) Amide I2866 ν-(CH)3296 ν-(OH)
50
Wavenumber (cm-1)
Appendix 2
500 MHz 1H NMR Spectra of 1% (w/w) solution of 75-85% deacetylated Chitosan in D2O/HCl
51
R = H or COCH3
1
2
3, 4, 5, 6
CH3
(Acetyl group)
Appendix 4
FT-IR spectra of [Ch-DTPA]
Wavenumber (cm-1) Peak interpretation87, 100
1034 ν-(C-O-C) Chitosan backbone1067 ν-(C-O-C) Chitosan backbone1159 ν-(C-O-C) Bridging oxygen1316 ν-(C-O-C)1400 δ-(CH2)1543 δ-(NH) Amide II1654 ν-(C=O) Amide I2920 ν-(CH)3304 ν-(OH)
52
Wavenumber (cm-1)
Appendix 4
500 MHz 1H NMR Spectra of 1% (w/w) solution of [Ch-DTPA] in D2O/HCl
53
1 3, 4, 5, 6
B and 2
CH3
(Acetyl group)
AC
D
R = H or COCH3
Appendix 5
FT-IR spectra of [Ch-EDTA]
Wavenumber (cm-1) Peak interpretation87, 100
1034 ν-(C-O-C) Chitosan backbone1067 ν-(C-O-C) Chitosan backbone1150 ν-(C-O-C) Bridging oxygen1316 ν-(C-O-C)1383 δ-(CH2)1560 δ-(NH) Amide II1630 ν-(C=O) Amide I2940 ν-(CH)3289 ν-(OH)
Appendix 6
500 MHz 1H NMR Spectra of 1% (w/w) solution of [Ch-EDTA] in D2O/HCl
54
Wavenumber (cm-1)
Appendix 7
Cross coupling reaction
55
B and 2
CH3
(Acetyl group)
R = H or COCH3
1 3, 4, 5, 6
C
A
D
In the first step EDC activates the carboxylic acid which forms an O-acylisourea
intermediate, which contains the newly formed O-acylisourea ester bond.101
The activated carboxylic acid group can then react with an amine group to form an amide
bond. However EDC lacks the efficiency to act as a crosslinking agent; the O-acylisourea
intermediate does not react fast enough with the amine group which results in the
hydrolysis of EDC and the regeneration of the Carboxyl group. Sulfo-NHS is used in
conjunction with EDC to increase the efficiency of this cross coupling, this is due to Sulfo-
NHS being less prone to hydrolysis and forming a more stable intermediate.97
Appendix 8
Synthesis of the modified chitosan polymers
56
Medium molecular weight chitosan (75-85% deacetylated) was used for the
synthesis of both Ch-DTPA and Ch-EDTA. This reaction is a slightly modified version of a
synthesis that had been defined previously (Scheme 1 and 2). 95
Scheme 2. Reaction scheme of DTPA-Ch. The DTPA-Ch monomer is based on
the theoretical assumption of a 50% loading of DTPA on chitosan.
Scheme 3. Reaction scheme of EDTA-Ch. The EDTA-Ch monomer is based on
the theoretical assumption of a 20% loading of EDTA on chitosan.
Appendix 9
Preparation of the modified chitosan polymers
57
Preparation of Chitosan-DTPA [Ch-DTPA]
A solution of chitosan (0.2g (2% weight)) was made in deionised water (10mL) (with
1% v/v of HCl 6M) and the pH was set to 4.7 using NaOH (1M) when the chitosan had
dissolved. DTPA (0.1g) was activated using a mixture of NHS ([NHS] / [COOH] = 1.3) and EDC
([EDC] / [COOH] = 1.3) in a buffer solution (pH 4.7). This was then added to the chitosan
solution and stirred for 24 hours at room temperature. The product was purified using
dialysis tubing against distilled water for 24 hours followed by lyophilisation to afford 0.248g
(83%) of product. Mw 697.7g mol-1 (based on one monomer of Ch-DTPA shown in Scheme
2)
Preparation of Chitosan-EDTA [Ch-EDTA]
A solution of chitosan (0.5g (5% weight)) was made in deionised water (25mL) (with
1% v/v of HCl 6M) and the pH was set to 4.7 using NaOH (1M) when the chitosan had
dissolved. EDTA (0.1g) was activated using a mixture of NHS ([NHS] / [COOH] = 1.3) and EDC
([EDC] / [COOH] = 1.3) in a buffer solution (pH 4.7). This was then added to the chitosan
solution and stirred for 24 hours at room temperature. The product was purified using
dialysis tubing against distilled water for 24 hours followed by lyophilisation to afford 0.227g
(76%) of product. Mw 1080g mol-1 (based on one monomer of Ch-EDTA shown in Scheme
3)
Appendix 10
Synthetic polymer-based macromolecular contrast agents65
macromolecular contrast
Chelate ty MW no.Gd(III
% Gdconten
ion r1(mM-1 s
mol. r1(m M-1
freq(MHz
T(°C
58
agent pe (Da) ) t 1)a,b
s-1)a ) )
polylysine-GdDTPA Gd-DTTAMA
48700 60-70 13.1 850 20 39
PL-GdDTPA Gd-DTTAMA
50000 10.8 10 37
PL-GdDTPA Gd-DTTAMA
23810
0
11.74 100 37
PL-GdDTPA Gd-DTTAMA
89900 11.58 100 37
PL-GdDTPA Gd-DTTAMA
56000 10.56 100 37
PL-GdDTPA Gd-DTTAMA
7700 11.67 100 37
PL-GdDOTA Gd-DO3AMA
65000 13.03 10 37
Gd-DTPA-PEG
Ipolylysine
Gd-DTTAMA
10800 6-7 9.64 6 39 20 37
Gd-DTPA-PEG
IIpolylysine
Gd-DTTAMA
13600 8-9 9.85 6 51 20 37
Gd-DTPA-PEG
IIIpolylysine
Gd-DTTAMA
18500 9-10 7.75 6 57 20 37
Gd-DTPA-PEG
IVpolylysine
Gd-DTTAMA
21900 11-12 8.42 6 69 20 37
Gd-DTPA-PEG
Vpolylysine
Gd-DTTAMA
31500 7-8 3.73 6 45 20 37
a Water.b Asterisk (*) indicates values estimated from NMRD curve.
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