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.

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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.

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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)

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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.

References

59

1Rana, B. P. S., The preparation and Evaluation of novel polymeric contrast agents, Cardiff Univesity, 2012.2 Chang; Raymond, Chemistry, 9 ed, McGraw-Hill, 2007, p 523 Atkins, P.; Paula, J., Atkins’ Physcial Chemistry, 8 ed.; Oxford University press, 2006, p 514-5534 Rosman, K. J. R.;Taylor, P. D. P., IUPAC Subcommittee for Isotopic Abundance Measurements, Pure Appl. Chem.

1999, 71, 1593-16075 Szumowski, J.; Plewes, D. B., Separation of lipid and water MR imaging signals by chopper averaging in the time

domain. Radiology, 1987, 165 (1), 247-2506 Brown, M. A.; Semelka, R. C., MRI: Basic Principles and Applications, 2 ed., John Wiley & Sons: 2011; p 71 Section 6-17 Farrar, T. C., Introduction to Pulse Nmr Spectroscopy. Farragut Pr: 1989; p 184

8 Gandhi, S. N.; Brown, M. A.; Wong, J. G.; Aguirre, D. A.; Sirlin, C. B., MR Contrast Agents for Liver Imaging: What,

When, How, RadioGraphics, 2006, 26, 1621-16369 Raymond, K. N.; Pierre. V. C., Next Generation, High Relaxivity Gadolinium MRI Agents, Bioconjugate Chem. 2005, 16,

3-8.10 Toth, E.; Helm, L.; Merbach, A., Relaxivity of MRI Contrast Agents, Contrast Agents I. Topics in Current Chemistry

2002, 221, 61-101.11 . Lauffer, R. B., Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and

design. Chemical Reviews 1987, 87 (5), 901-92.12 Li, Z. et al. The gadolinium complexes with polyoxometalates as potential MRI contrast agents. Magnetic Resonance

Imaging 2007, 25, 412–41713 Sosnovik, D. E.; Weissleder, R., Emerging concepts in molecular MRI. Current Opinion in Biotechnology 2007, 18 (1),

4-10.14 Zhang, Z.; Kolodziej, A. F.; Greenfield, M. T.; Caravan, P., Heteroditopic Binding of Magnetic Resonance Contrast

Agents for Increased Relaxivity. Angewandte Chemie International Edition 2011, 50 (11), 2621-2624.15 Merbach, A. E.; Toth, E., The chemistry of contrast agents in medical magnetic resonance imaging. Wiley: 2001; p

45-119.16 Strandberg, E.; Westlund, P.-O., 1H NMRD Profile and ESR Lineshape Calculation for an Isotropic Electron Spin

System withS= 7/2. A Generalized Modified Solomon–Bloembergen– Morgan Theory for Nonextreme Narrowing

Conditions. Journal of Magnetic Resonance, Series A 1996, 122 (2), 179-191.17 Peters, J. A.; Huskens, J.; Raber, D. J., Lanthanide induced shifts and relaxation rate enhancements. Progress in

Nuclear Magnetic Resonance Spectroscopy 1996, 28 (3–4), 283- 350.18 Alpoim, M. C.; Urbano, A. M.; Geraldes, C. F. G. C.; Peters, J. A., Determination of the number of inner-sphere water

molecules in lanthanide(III) polyaminocarboxylate complexes. Journal of the Chemical Society, Dalton Transactions

1992, (3), 463-467.19 Djanashvili, K.; Peters, J. A., How to determine the number of inner-sphere water molecules in Lanthanide(III)

complexes by 17O NMR spectroscopy. A technical note. Contrast Media & Molecular Imaging 2007, 2 (2), 67-71.20 Zech, S. G.; Sun, W.-C.; Jacques, V.; Caravan, P.; Astashkin, A. V.; Raitsimring, A. M., Probing the Water Coordination

of Protein-Targeted MRI Contrast Agents by Pulsed ENDOR Spectroscopy. ChemPhysChem 2005, 6 (12), 2570-2577.21 Raitsimring, A. M.; Astashkin, A. V.; Baute, D.; Goldfarb, D.; Poluektov, O. G.; Lowe, M. P.;

Zech, S. G.; Caravan, P., Determination of the Hydration Number of Gadolinium(III) Complexes by High-Field Pulsed

17O ENDOR Spectroscopy. ChemPhysChem 2006, 7 (7), 1590-1597.22 Horrocks, W. D.; Sudnick, D. R., Lanthanide ion probes of structure in biology. Laser-induced luminescence decay

constants provide a direct measure of the number of metal-coordinated water molecules. Journal of the American

Chemical Society 1979, 101 (2), 334-340.23 Beeby, A.; M. Clarkson, I.; S. Dickins, R.; Faulkner, S.; Parker, D.; Royle, L.; S. de Sousa, A.; A. Gareth Williams, J.;

Woods, M., Non-radiative deactivation of the excited states of europium, terbium and ytterbium complexes by

proximate energy-matched OH, NH and CH oscillators: an improved luminescence method for establishing solution

hydration states. Journal of the Chemical Society, Perkin Transactions 2 1999, (3), 493-504.24 Supkowski, R. M.; Horrocks Jr, W. D., On the determination of the number of water molecules, q, coordinated to

europium(III) ions in solution from luminescence decay lifetimes. Inorganica Chimica Acta 2002, 340 (0), 44-48.25 Aime, S.; Fasano, M.; Terreno, E., Lanthanide(III) chelates for NMR biomedical applications. Chemical Society

Reviews 1998, 27 (1), 19-29.26 Woods, M.; Botta, M.; Avedano, S.; Wang, J.; Sherry, A. D., Towards the rational design of

MRI contrast agents: a practical approach to the synthesis of gadolinium complexes that exhibit optimal water

exchange. Dalton Transactions 2005, (24), 3829-3837.27 Hermann, P.; Kotek, J.; Kubiček, V.; Lukeš, I., Gadolinium(III) complexes as MRI contrast agents: ligand design and

properties of the complexes. Dalton Transactions 2008, (23), 3027–304728 Nicolle, G. M.; Toth, E.; Schmitt-Willich, H.; Raduchel, B.; Merbach, A. E., The Impact of Rigidity and Water Exchange

on the Relaxivity of a Dendritic MRI Contrast Agent. Chemistry – A European Journal 2002, 8 (5), 1040-1048.29 Toth, E.; Pubanz, D.; Vauthey, S.; Helm, L.; Merbach, A. E., The Role of Water Exchange in Attaining Maximum

Relaxivities for Dendrimeric MRI Contrast Agents. Chemistry – A European Journal 1996, 2 (12), 1607-1615.30 Wiener, E.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C., Dendrimer-

based metal chelates: A new class of magnetic resonance imaging contrast agents. Magnetic Resonance in Medicine

1994, 31 (1), 1-8.31 Eisinger, J.; Shulman, R. G.; Blumberg, W. E., Relaxation Enhancement by Paramagnetic Ion Binding in

Deoxyribonucleic Acid Solutions. Nature 1961, 192 (4806), 963-964.32 Lauffer, R. B.; Brady, T. J., Preparation and Water Relaxation Properties of Proteins Labeled with Paramagnetic Metal

Chelates. Magnetic Resonance Imaging 1985, 3, 11-1633 Kielar, F.; Tei, L.; Terreno, E.; Botta, M., Large Relaxivity Enhancement of Paramagnetic Lipid Nanoparticles by

Restricting the Local Motions of the GdIII Chelates. Journal of the American Chemical Society 2010, 132 (23), 7836-

7837.34 Xu, Q.; Zhu, L.; Yu, M.; Feng, F.; An, L.; Xing, C.; Wang, S., Gadolinium(III) chelated conjugated polymer as a potential

MRI contrast agent. Polymer, 2010, 51, 1336–1340.35 Tekade, R. K.; Kumar, P. V.; Jain, N. K., Dendrimers in Oncology: An Expanding Horizon. Chemical Reviews 2008, 109

(1), 49-87.36 Peters, J. A., Glycoconjugate probes and targets for molecular imaging using magnetic resonance. Future Med.

Chem. 2010 2(3), 409–425

37 Lebduskova, P.; Hermann, P.; Helm, L.; Toth, E.; Kotek, J.; Binnemans, K.; Rudovsky, J.; Lukes, I.; Merbach, A. E.,

Gadolinium(iii) complexes of mono- and diethyl esters of monophosphonic acid analogue of DOTA as potential MRI

contrast agents: solution structures and relaxometric studies. Dalton Transactions 2007, (4), 493-501.38 Rudovsky, J.; Botta, M.; Hermann, P.; Hardcastle, K. I.; Lukeš, I.; Aime, S., PAMAM Dendrimeric Conjugates with a

Gd−DOTA Phosphinate Derivative and Their Adducts with Polyaminoacids: The Interplay of Global Motion, Internal

Rotation, and Fast Water Exchange. Bioconjugate Chemistry 2006, 17 (4), 975-987.39 Merbach, A. S., Helm, L., Toth, E., the chemistry of contrast agents in medical magnetic resonance imaging, 8 ed,

Wiley, 2013, 3740 Aime, S.; Botta, M.; Fasano, M.; Geninatti Crich, S.; Terreno, E., 1H and 17O-NMR relaxometric investigations of

paramagnetic contrast agents for MRI. Clues for higher relaxivities. Coordination chemistry reviews 1999, 185–186 (0),

321-333.41 Xu, J.; Franklin, S. J.; Whisenhunt, D. W.; Raymond, K. N., Gadolinium complex of tris[(3- hydroxy-1-methyl- 2-oxo-

1,2-didehydropyridine-4-carboxamido)ethyl]-amine: A New Class of gadolinium magnetic resonance relaxation agents.

Journal of the American Chemical Society 1995, 117 (27), 7245-7246.42 Yang, C.; Chuang, K., Gd(III) chelates for MRI contrast agents: from high relaxivity to ‘‘smart’’, from blood pool to

blood–brain barrier permeable. Med. Chem. Commun. 2012, 3, 552.43 Bryson, J. M.; Chu, W.-J.; Lee, J.-H.; Reineke, T. M., A β-Cyclodextrin “Click Cluster” Decorated with Seven

Paramagnetic Chelates Containing Two Water Exchange Sites. Bioconjugate Chemistry 2008, 19 (8), 1505-1509.44 Jebasingh, B.; Alexander, V., Synthesis and Relaxivity Studies of a Tetranuclear Gadolinium(III) Complex of DO3A as a

Contrast-Enhancing Agent for MRI. Inorganic Chemistry 2005, 44 (25), 9434 9443.

45Cheng, S. G., Musculoskeletal MRI: Contrast and non-contrast applications. Applied Radiology 2002, 31(6), 81-86

46 Bjørnerud, A.; Johansson, L., The utility of superparamagnetic contrast agents in MRI: theoretical consideration and

applications in the cardiovascular system. NMR Biomed. 2004, 17, 465–477.47 Carr, D. H.; and Gadian, D. G., Contrast Agents in Magnetic Resonance Imaging. ClinicalRadiology, 1985, 36, 561-

568.48 Olsson, M. B. E.; Persson, B. R. B.; Salford, L. G.; Schroder, U., Ferromagnetic particles as contrast agent in T2 NMR

imaging. Magnetic Resonance Imaging 1986, 4 (5), 437-440.49 Kehagias, D. T.; Gouliamos, A. D.; Smyrniotis, V.; Vlahos, L. J., Diagnostic efficacy and safety of MRI of the liver with

superparamagnetic iron oxide particles (SH U 555 A). Journal of Magnetic Resonance Imaging 2001, 14 (5), 595-601.50 Thunus, L.; Lejeune, R., Overview of transition metal and lanthanide complexes as diagnostic tools. Coordination

chemistry reviews 1999, 184 (1), 125-155.51 Mendonca-Dias, M. H.; Gaggelli, E.; Lauterbur, P. C., Paramagnetic contrast agents in nuclear magnetic resonance

medical imaging. Seminars in Nuclear Medicine 1983, 13 (4), 364-376.52 Weinmann, H.; Brasch, R.; Press, W.; Wesbey, G., Characteristics of gadolinium-DTPA complex: a potential NMR

contrast agent. American Journal of Roentgenology 1984, 142 (3), 619-624.53 Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B., Gadolinium(III) Chelates as MRI Contrast Agents: Structure,

Dynamics, and Applications. Chemical Reviews 1999, 99 (9), 2293-2352.54 Grobner, T., Gadolinium – a specific trigger for the development of nephrogenic fibrosing dermopathy and

nephrogenic systemic fibrosis? Nephrology Dialysis Transplantation 2006, 21 (4), 1104-1108.

55 Marckmann, P.; Skov, L.; Rossen, K.; Dupont, A.; Damholt, M. B.; Heaf, J. G.; Thomsen, H. S., Nephrogenic Systemic

Fibrosis: Suspected Causative Role of Gadodiamide Used for Contrast- Enhanced Magnetic Resonance Imaging. Journal

of the American Society of Nephrology 2006, 17 (9), 2359-2362.

56 Thomsen, H. S., Nephrogenic Systemic Fibrosis: History and Epidemiology. Radiologic Clinics of North America

2009, 47(5), 827–831.57 Broome, D., Nephrogenic systemic fibrosis associated with gadolinium based contrast agents: A summary of the

medical literature reporting. European Journal of Radiology 2008, 6, 230–234.58 Drahoš, B.; Kotek, J.; Hermann, P.; Lukeš, I.; Toth, E. v., Mn2+ Complexes with Pyridine- Containing 15-Membered

Macrocycles: Thermodynamic, Kinetic, Crystallographic, and 1H/17O Relaxation Studies. Inorganic Chemistry 2010, 49

(7), 3224-3238.59 Aschner, M.; Aschner, J. L., Manganese neurotoxicity: Cellular effects and blood-brain barrier transport.

Neuroscience & Biobehavioral Reviews 1991, 15 (3), 333-340.60 Gallez, B.; Baudelet, C.; Adline, J.; Geurts, M.; Delzenne, N., Accumulation of Manganese in the Brain of Mice after

Intravenous Injection of Manganese-Based Contrast Agents. Chemical Research in Toxicology 1997, 10 (4), 360-363.61 Olanow, C. W., Manganese-Induced Parkinsonism and Parkinson's Disease. Annals of the New York Academy of

Sciences 2004, 1012 (1), 209-223.62 Troughton, J. S.; Greenfield, M. T.; Greenwood, J. M.; Dumas, S.; Wiethoff, A. J.; Wang, J.; Spiller, M.; McMurry, T. J.;

Caravan, P., Synthesis and Evaluation of a High Relaxivity Manganese(II)-Based MRI Contrast Agent. Inorganic

Chemistry 2004, 43 (20), 6313-6323.63 Small, W. C.; DeSimone-Macchi, D.; Parker, J. R.; Sukerkar, A.; Hahn, P. F.; Rubin, D. L.; Zelch, J. V.; Kuhlman, J. E.;

Outwater, E. K.; Weinreb, J. C.; Brown, J. J.; de Lange, E. E.; Woodward, P. J.; Arildsen, R.; Foster, G. S.; Runge, V. M.;

Aisen, A. M.; Muroff, L. R.; Thoeni, R. F.; Parisky, Y. R.; Tanenbaum, L. N.; Totterman, S.; Herfkens, R. J.; Knudsen, J.;

Laster, J. R. E.; Duerinckx, A.; Stillman, A. E.; Spritzer, C. E.; Saini, S.; Rofsky, N. M.; Bernardino, M. E., A multisite phase

III study of the safety and efficacy of a new manganese chloride-based gastrointestinal contrast agent for MRI of the

abdomen and pelvis. Journal of Magnetic Resonance Imaging 1999, 10 (1), 15-24.64 Pan, D.; Caruthers, S. D.; Senpan, A.; Schmieder, A. H.; Wickline, S. A.; Lanza, G. M., Revisiting an old friend:

manganese-based MRI contrast agents. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2011,

3 (2), 162-173.65 Rocklage, S. M.; Cacheris, W. P.; Quay, S. C.; Hahn, F. E.; Raymond, K. N., Manganese(II) N,N'-

dipyridoxylethylenediamine-N,N'-diacetate 5,5'-bis(phosphate). Synthesis and characterization of a paramagnetic

chelate for magnetic resonance imaging enhancement. Inorganic Chemistry 1989, 28 (3), 477-485.66 Elizondo, G.; Fretz, C. J.; Stark, D. D.; Rocklage, S. M.; Quay, S. C.; Worah, D.; Tsang, Y. M.; Chen, M. C.; Ferrucci, J. T.,

Preclinical evaluation of MnDPDP: new paramagnetic hepatobiliary contrast agent for MR imaging. Radiology 1991,

178 (1), 73-78.67 Mahmoudi, M.; Sant, S.; Wang, B.; Laurent, S.; Sen, S., Superparamagnetic iron oxide nanoparticles (SPIONs):

Development, surface modification and applications in chemotherapy. Advanced Drug Delivery Reviews 2011, 63 (1–

2), 24–46. 68 Clarkson, R., Blood-Pool MRI Contrast Agents: Properties and Characterization Contrast Agents I. 2002, 221, 201-

235.

69 Duarte, M. G.; Gil, M. H.; Peters, J. A.; Colet, J. M.; Elst, L. V.; Muller, R. N.; Geraldes, C. F. G. C., Synthesis,

Characterization, and Relaxivity of Two Linear Gd(DTPA)−Polymer Conjugates. Bioconjugate Chemistry 2001, 12 (2),

170-177.70 Brasch, R. C., Rationale and applications for macromolecular Gd-based contrast agents. Magnetic Resonance in

Medicine 1991, 22 (2), 282-287.71 Schuhmann-Giampieri, G. ; Schmitt-Willich, H.; Frenzel, T.; PRESS, W. R.; Weinmann, H. J., In Vivo and In Vitro

Evaluation of Gd-DTPA-Polylysine as a Macromolecular Contrast Agent for Magnetic Resonance Imaging. Investigative

Radiology 1991, 26 (11), 969-974.72 Bogdanov Jr, A. A.; Weissleder, R.; Brady, T. J., Long-circulating blood pool imaging agents. Advanced Drug Delivery

Reviews 1995, 16 (2–3), 335-348.73 . Prasuhn, J. D. E.; Yeh, R. M.; Obenaus, A.; Manchester, M.; Finn, M. G., Viral MRI contrast agents: coordination of

Gd by native virions and attachment of Gd complexes by azidealkyne cycloaddition. Chemical Communications 2007,

(12), 1269-1271.74 Allen, M.; Bulte, J. W. M.; Liepold, L.; Basu, G.; Zywicke, H. A.; Frank, J. A.; Young, M.; Douglas, T., Paramagnetic viral

nanoparticles as potential high-relaxivity magnetic resonance contrast agents. Magnetic Resonance in Medicine 2005,

54 (4), 807-812.75 Anderson, E. A.; Isaacman, S.; Peabody, D. S.; Wang, E. Y.; Canary, J. W.; Kirshenbaum, K., Viral Nanoparticles

Donning a Paramagnetic Coat: Conjugation of MRI Contrast Agents to the MS2 Capsid. Nano Letters 2006, 6 (6), 1160-

1164.76 Hooker, J. M.; Datta, A.; Botta, M.; Raymond, K. N.; Francis, M. B., Magnetic Resonance Contrast Agents from Viral

Capsid Shells: A Comparison of Exterior and Interior Cargo Strategies. Nano Letters 2007, 7 (8), 2207-2210.77 Lauffer, R. B.; Brady, T. J., Preparation and water relaxation properties of proteins labelled with paramagnetic metal

chelates. Magnetic Resonance Imaging 1985, 3 (1), 11-16.78 Zech, S. G.; Eldredge, H. B.; Lowe, M. P.; Caravan, P., Protein Binding to Lanthanide(III) Complexes Can Reduce the

Water Exchange Rate at the Lanthanide. Inorganic Chemistry

2007, 46 (9), 3576-3584.79 Aime, S.; Botta, M.; Fasano, M.; Crich, S. G.; Terreno, E., Gd(III) complexes as contrast agents for magnetic resonance

imaging: a proton relaxation enhancement study of the interaction with human serum albumin. Journal of Biological

Inorganic Chemistry 1996, 1 (4), 312-319.80 Muller, R. N.; Raduchel, B.; Laurent, S.; Platzek, J.; Pierart, C.; Mareski, P.; Vander Elst, L., Physicochemical

Characterization of MS-325, a New Gadolinium Complex, by Multinuclear Relaxometry. European Journal of Inorganic

Chemistry 1999, 1999 (11), 1949-1955.81 Doble, D. M. J.; Botta, M.; Wang, J.; Aime, S.; Barge, A.; Raymond, K. N., Optimization of the Relaxivity of MRI

Contrast Agents: Effect of Poly(ethylene glycol) Chains on the Water- Exchange Rates of GdIII Complexes. Journal of

the American Chemical Society 2001, 123 (43), 10758-10759.82 Eweis, M.; Elkholy, S. S.; Elsabee, M. Z., Antifungal efficacy of chitosan and its thiourea derivatives upon the growth

of some sugar-beet pathogens. International Journal of Biological Macromolecules 2006, 38 (1), 1-8.

91. Chen, S.; Wu, G.; Zeng, H., Preparation of high antimicrobial activity thiourea chitosan–Ag+ complex. Carbohydrate

Polymers 2005, 60 (1), 33-38.

83 Zhong, Z.; Xing, R.; Liu, S.; Wang, L.; Cai, S.; Li, P., Synthesis of acyl thiourea derivatives of chitosan and their

antimicrobial activities in vitro. Carbohydrate Research 2008, 343 (3), 566- 570.84 Zheng, Y.; Yi, Y.; Qi, Y.; Wang, Y.; Zhang, W.; Du, M., Preparation of chitosan–copper complexes and their antitumor

activity. Bioorganic & Medicinal Chemistry Letters 2006, 16 (15), 4127-4129.85 Mekahlia, S.; Bouzid, B., Chitosan-Copper (II) complex as antibacterial agent: synthesis, characterization and

coordinating bond- activity correlation study. Physics Procedia 2009, 2 (3), 1045-1053.86 Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A., Typical Physicochemical Behaviors of Chitosan in Aqueous

Solution. Biomacromolecules 2003, 4 (3), 641-648.87 Kasaai, M. R., A review of several reported procedures to determine the degree of Nacetylation for chitin and

chitosan using infrared spectroscopy. Carbohydrate Polymers 2008, 71 (4), 497-508.88 Rinaudo, M., Chitin and chitosan: Properties and applications. Progress in Polymer Science 2006, 31 (7), 603-632.89 Ravi Kumar, M. N. V., A review of chitin and chitosan applications. Reactive and Functional Polymers 2000, 46 (1), 1-

27.90 Zhuangdong, Y., Study on the synthesis and catalyst oxidation properties of chitosan bound nickel (II) complexes. J

Agric Food Chem 2007, 21 (5), 22-24.91 Rong Huei, C.; Hwa, H.-D., Effect of molecular weight of chitosan with the same degree of deacetylation on the

thermal, mechanical, and permeability properties of the prepared membrane. Carbohydrate Polymers 1996, 29 (4),

353-358.92 Yomota, C.; Miyazaki, T.; Okada, S., Determination of the viscometric constants for chitosan and the application of

universal calibration procedure in its gel permeation chromatography. Colloid & Polymer Science 1993, 271 (1), 76-82.93 Chen, R. H.; Tsaih, M. L., Effect of temperature on the intrinsic viscosity and conformation of chitosans in dilute HCl

solution. International Journal of Biological Macromolecules 1998, 23 (2), 135-141.94 Huang, M.; Huang, Z. L.; Bilgen, M.; Berkland, C., Magnetic resonance imaging of contrastenhanced polyelectrolyte

complexes. Nanomedicine : nanotechnology, biology, and medicine 2008, 4 (1), 30-40.95 Darras, V.; Nelea, M.; Winnik, F. M.; Buschmann, M. D., Chitosan modified with gadolinium

diethylenetriaminepentaacetic acid for magnetic resonance imaging of DNA/chitosan nanoparticles. Carbohydrate

Polymers 2010, 80 (4), 1137-1146.96 Justi, K. C.; Favere, V. T.; Laranjeira, M. C. M.; Neves, A.; Casellato, A., Synthesis and Characterization of Modified

Chitosan Through Immobilization of Complexing Agents. Macromolecular Symposia 2005, 229 (1), 203-207.97 Chung, T. W.; Yang, J.; Akaike, T.; Cho, K. Y.; Nah, J. W.; Kim, S. I.; Cho, C. S., Preparation of alginate/galactosylated

chitosan scaffold for hepatocyte attachment. Biomaterials 2002, 23 (14), 2827-2834.98 Feng, J.; Sun, G.; Pei, F.; Liu, M., Comparison between GdDTPA and two gadolinium polyoxometalates as potential

MRI contrast agents. Journal of Inorganic Biochemistry 2002, 92 (3–4), 193-199.99 Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B., Gadolinium(III) Chelates as MRI Contrast Agents: Structure,

Dynamics, and Applications. Chemical Reviews 1999, 99 (9), 2293-2352.100 Duarte, M. L.; Ferreira, M. C.; Marvao, M. R.; Rocha, J., An optimised method to determine the degree of

acetylation of chitin and chitosan by FTIR spectroscopy. International Journal of Biological Macromolecules 2002, 31

(1–3), 1-8.101 Nakajima, N.; Ikada, Y., Mechanism of Amide Formation by Carbodiimide for Bioconjugation in Aqueous Media.

Bioconjugate Chemistry 1995, 6 (1), 123-130.