Towards the Development of the Dual Modal Contrast Agent for Computed Tomography and Ultrasound

DEGREE PROJECT IN MEDICAL ENGINEERING, SECOND CYCLE, 30 CREDITS STOCKHOLM, SWEDEN 2016

HONGJIAN CHEN

KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF TECHNOLOGY AND HEALTH

Abstract

Nowadays hybrid imaging modalities are new trends in medical imaging. To improve the diagnostic outcome of hybrid imaging, multimodal contrast agents need to be developed. For example, hybrid contrast agents for computer tomography and ultrasound (CACTUS) are one of those desirable hybrid contrast agents for the modern medical imaging.

Polyvinyl alcohol (PVA) micro-bubbles (MBs) are one of the latest generations of ultrasound contrast agents (UCAs). PVA MBs are more stable and offer longer circulation and on-shelf storage time compare to other UCAs. However, the current use as contrast agent is limited only to ultrasound imaging.

In this project, we fabricated and characterized hybrid contrast agents based on PVA MBs.

Two methods for developing hybrid contrast agents were proposed. The first method is to combine MBs, currently used as an ultrasound contrast agent, with gold nanoparticles that are used as a preclinical contrast agent for computer tomography (CT). The second method is to determine at which concentration plain MBs suspension has both considerable negative contrast in CT and enhancement of the backscattered signal in ultrasound imaging.

Both methods were evaluated and optimized. A scenario to achieve promising hybrid contrast agent was described in this report.

Keywords

Computed tomography, Ultrasound, Hybrid contrast agents, Polyvinyl alcohol

Table of Contents

Introduction ...... 1

Method ...... 2

Result ...... 3

Microscopy test ...... 3

Plain MB and low gold-embedded MB ...... 3

High gold-embedded MBs ...... 5

Ultrasound test ...... 7

Ultrasound calibration ...... 7

Evaluation of the backscattered efficiency...... 10

CT test ...... 12

Discussion ...... 15

Microscopy test ...... 15

Ultrasound test ...... 16

CT test ...... 18

Conclusion ...... 21

Acknowledgment ...... 22

References ...... 23

Appendix ...... 24

Introduction

Contrast agents improve the diagnostic values of various imaging modalities. Most of the current contrast agents are specifically designed and for a particular image modality. There is a desire, however, for new hybrid contrast agents for the hybrid imaging modalities.

Usually the hybrid contrast agents are the combination of multiple contrast agents, which have been already used in clinical or preclinical studies. For example, Kim, D. et al. [1] combined iron oxide, gold nanoparticles, and amphiphilic polymer to fabricate a computer tomography (CT)/ Magnetic Resonance Imaging (MRI) hybrid contrast agent.

In this project, hybrid contrast agents for CT and ultrasound (CACTUS) were developed based on polyvinyl alcohol (PVA) micro-bubble (MB). Two methods for fabricating hybrid contrast agent were proposed.

For hybrid contrast agents with high X-ray mass absorption coefficient, gold nanoparticles were added during the fabrication of the plain MB.

PVA MBs are one of the latest generations of ultrasound contrast agents. A previous study [2] shows that the PVA shell is able to incorporate pharmacological material chemically or mechanically, for instance iron oxide and nitric oxide. Gold nanoparticle is a contrast agent for CT and is currently used in preclinical studies and translational research.

The plain MBs can potentially work as a hybrid contrast agent with low X-ray mass absorption coefficient for ultrasound imaging and CT due to its gas core, which has a significant smaller mass absorption than the tissue.

Here, we will present the fabrication and characterization of the two types of MBs and assess the feasibility of using those MBs as hybrid contrast agent for CT and ultrasound imaging.

Method

The PVA MB is a hollow sphere. The core of the sphere contains air that is encapsulated by the PVA shell. The shell is formed by the polymerization reaction of PVA. In this study, gold nanoparticles were proposed to be added during the polymerization. It is expected that some of the gold nanoparticles will be trapped inside the PVA shell. The structures of the plain and the gold-embedded MB are shown in figure 1.

Figure1. The structure of the MB

MB and gold-embedded MB are fabricated based on the previous developed protocol [3].

Materials:

 Polyvinyl alcohol (Sigma-Aldrich, St.Louis, USA)  ExiTronTM nano 12000(gold nano particles Miltenyi Biotec Inc, Bergisch Gladbach, Germany)

 NaIO4 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)

Procedure:

The fabrication procedure has two major aspects: suspension preparation and washing.

In the suspension preparation step, 4g PVA powder was mixed with 200ml of Milli-Q water in a 600ml beaker and heated to 80℃ under high agitation. Then, 380mg of NaIO4 was added into the mixture together with 0, 100 and 555 µl of ExiTronTM nano 12000 for the generation of plain MBs, low gold-embedded MBs and high gold-embedded MBs, respectively. The mixtures were kept at 80℃ for 1 hour and subsequently cooled to room temperature by immersing the beaker in an ice-water bath. In order to form the PVA shell, ultrathurrax motor (IKA Works GmbH & Co Staufen, Germany) was set as 8000 revolutions/min, at room temperature for 2 hours. Finally, a separation funnel was used to wash the MBs suspension 10 times with 24 hours’ intervals.

After fabrication, the size, ultrasonic feature and X-ray mass absorption coefficient were characterized. 3

Result

Microscopy test

Plain MB and low gold-embedded MB

The size and concentration of the MBs were observed by microscope ECLIPSE Ci-S (Nikon, Tokyo, Japan). Samples were prepared by diluting MBs suspension 20 times. A 20X objective was selected to acquire the images. Figure 2 is an example of the acquired images. The MBs are black, which shows the high optical contrast of the MBs.

Figure 2. MBs under X20 objective lens

The images acquired by the microscope were processed by the ImageJ (Version 1.50b developed by Wayne Rasband, National institutes of health, USA). An example of the processed images is shown in Figure 3. 4

Figure 3. Processed image of MBs under X20 objective lens

8 images of each sample in different positions were acquired and processed.

The area values of the individual MBs are acquired. Then, the diameters of those individual MBs can be obtained using equation 1:

푑 = 2푅 = 2√퐴푟푒푎 푣푎푙푢푒 (1) 휋

The concentration of the MBs suspension can be obtained using equation 2:

푐표푛푐푒푛푡푟푎푡푖표푛 = 퐴푣푒푟푎푔푒 푛푢푚푏푒푟 표푓 푀퐵푠 푖푛 푠푎푚푝푙푒 푎푟푒푎 푋 푑푖푙푢푡푖표푛 푓푎푐푡표푟 (2) 푉표푙푢푚푒 표푓 푠푎푚푝푙푒 푎푟푒푎

The size distribution and concentration of the Plain MBs and low gold-embedded MBs are summarized in the Table 1:

Table1. Distribution and concentration of the MBs

Sequence Type of the Mean diameter (um) Concentration (ml-1) bubbles 1 low 4.17±1.09 1.61*105 gold-embedded MBs 2 Plain MBs 3.56±1.08 1.84*108 5

Figure 4 and 5 represent the normalized frequency and the normalized Gaussian distribution of the size of the MBs.

0.25

0.2

0.15

0.1

0.05

Normalized frequency Normalized 0 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 Diameter of the MBs (um)

Normalized frequency Normalized Gauss Ditribution

Figure 4. Normalized frequency and the normalized Gaussian distribution of low gold-embedded MBs

0.25

0.2

0.15

0.1

0.05 Normalized frequency Normalized 0 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 Diameter of the MBs (um)

Normlized frequency Normalized Gauss Distribution

Figure 5. Normalized frequency and the normalized Gaussian distribution of plain MBs

High gold-embedded MBs

High gold-embedded MBs were produced following the same procedure, but a 5 times higher amount of gold nanoparticles was added. Unfortunately, microscopy imaging reveals that gas filled MBs were not formed, i.e. no bubbles were observed near the top of the counting chamber. However, broken parts of the MBs or water filled MBs were found near the bottom of the counting chamber. The water filled MBs are shown in light color due to the low optical contrast between the bubble and surrounding media. 6

Figure 6. Image of broken parts of the MBs or water filled MBs

Ultrasound test

Ultrasound calibration

A hydrophone set-up (Onda Corporation, Sunnyvale, USA) and transducer (Olympus NDT, Ville de Québec, Canada) were used to perform the ultrasound test. Before the characterization of the ultrasound response from the MBs, a calibration was performed to find the correspondence between the electrical output of the system and the corresponding acoustic parameters of the pressure field, produced by the transducer of the system.

During the calibration, the transducer and the hydrophone were immersed in a tank filled with degassed and deionized water. The flat transducer with a nominal frequency f = 3.5 MHz and diameter of half inch was used to produce a pressure field. The transducer was hold vertically in the water. The receiver scanned the horizontal plane, which is 80 cm (close to the near field) away from the transducer, under different electrical outputs. The peak negative pressure (PNP), mechanical index, pulse average intensity, and temporal average intensity of the point with highest negative pressure were recorded.

The characters of the pressure field are shown in Figure 7, 8, 9, and 10.

0.35

0.3

0.25

0.2

0.15 PNP(MPa) 0.1

0.05

0 0 5 10 15 20 25 Attenuation (dB)

Figure 7. Relation between PNP and the electrical output from the machine 8

0.07 0.06 0.05 0.04 0.03

0.02 Mechanical Mechanical index 0.01 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 PNP(MPa)

Figure 8. Relation between PNP and the mechanical index

18 16 14

12 intensity 10 8

(W/cm^2) 6 4

Pulse average average Pulse 2 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 PNP(MPa)

Figure 9. Relation between PNP and the pulse average intensity

20 intensity 15

10 (mW/cm^2)

5 Temporal average average Temporal 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 PNP(MPa)

Figure 10. Relation between PNP and the temporal average intensity 9

The power spectra of the excitation signal was constructed for each electrical output. The ratio between the amplitude of the fundamental and 2nd, 3rd, and 4th harmonic at each electrical output were calculated and presented in the Figure 11.

A B C

0 0 -10 0 -20 -10 -10 -30 -20 -20 -40 -30 -50 -40 -30 -60 -50 -40 -70 -60 -50 -80 -70 -90 -80 -60 0 5 10 15 20

0 5 10 15 20 0 5 10 15 20

dB dB MHz dB MHz D MHz

0 PNP(MPa) 0 0 .0 5 0 .1 0 .1 5 0 .2 0 .2 5 0 .3 0 .3 5 -5

-10

-15

-20 (dB) -25

-30

-35

-40 Ratio between fundamental and harmonic response fundamentalbetween harmonic andRatio

-45

Figure 11. (A), (B) and (C) are power spectra of the acoustic field at PNP 0.29, 0.16, 0.05 MPa. (D) is ratio between amplitude

of the fundamental and 2nd 3rd, and 4th harmonic at every level of PNP tested in this study. The blue, orange, and grey line

presents the 2nd harmonic, 3rd harmonic, and 4th harmonic respectively.

Evaluation of the backscattered efficiency

After the calibration, the Advanced measurement system model SNAP-Mark4-0.05-7-5kW (RITEC Inc, Warwick, USA) was used to evaluate the backscattered efficiency of the MBs suspension with various MBs concentrations. The MBs suspension was diluted to the concertation of 107, 5*106, and 106 ml-1. Then, the diluted MBs suspension was injected into a chamber. After that, the system was set up as shown in Figure 12. The transducer started to produce the pressure field. At last, the attenuations of MBs suspension with various concentrations were recorded and compared to the attenuation of water.

Transduce

Ultrasound beam

Sample

Reflector

Figure12. Schematic diagram of the experimental set-up

The Figure 13 presents the attenuation coefficient between the water and the MBs suspension at fundamental frequency, 2nd, and 3rd harmonics. The MBs suspension with a concentration of 107 ml-1 has the highest attenuation coefficient in all frequencies tested in this study. While the MBs suspension with concentration of 106 ml-1 has the lowest attenuation coefficient. All experiments were done at room temperature and the temperature difference of each experiment was within 2℃ (18±1℃). 11

Figure 13. Attenuation coefficients of the MBs suspension with respect to water in fundamental frequency, 2nd, and 3rd harmonic. The red line represents the MBs suspension with concertation of 107 ml-1. The magenta line represents the MBs

suspension with concentration of 5*106 ml-1. The blue line represents the MBs suspension with concentration of 106 ml-1

CT test

The in-house micro-CT developed at School of Health and Technology, Royal Institute of Technology, Flemingsberg, was used to do the CT test. It has a detector consisting of a 2400 × 2400 photon sensor array. Each photon sensor counts the number of photons to be captured. Finally, a 2400 * 2400 pixels image is acquired. The number of each pixel represent the number of photon captured by the corresponding sensor.

Two groups of experiments were performed.

In the first group of experiments, water, gold nanoparticles, plain MBs suspension, and low gold-embedded MBs suspension were loaded on a microplate. The microplate was placed on the detector. X-rays with 600uA current, voltage of 55 kV, and 90 kV were applied to the test. A filter was used to stop low energy photons when a voltage of 90 kV was applied. Images with exposure time of 500ms were acquired. The linear attenuation coefficient was determined using formula 3,

푆푎푚푝푙푒−퐷퐶 − ln 휇 = 퐿퐹−퐷퐶 (3) 푥 where Sample is the photon flux in case there is a sample between X-ray source and photon counters, LF is the photon flux for the case that there is no sample between X-ray source and photon counters, DC is the value photon counters read when there is no photon flux and X is the depth of the sample.

The contrast is determined by formula 4, which is adapted from Nebuloni et al. [4],

휇 −휇 contrast = 100 ∗ 푠푎푚푝푙푒 푤푎푡푒푟 (4) 휇푤푎푡푒푟

where 휇푠푎푚푝푙푒 is the average linear attenuation coefficient of the sample. The 휇푤푎푡푒푟 is the average linear attenuation coefficient in water.

Figure 14 is an image of test samples acquired by CT in the first group of experiments. Only the points inside the red circle are processed. 13

Figure 14. An image of test samples with X-ray of voltage 55kV, current 600uA, and exposure time 500ms

The results are summarized in the Table 2. When X-rays with a voltage of 55kV is applied, the plain MBs suspension has the highest contrast and the Low gold-embedded MBs suspension has the lowest contrast. When X-rays with a voltage of 90kV are applied, the contrast of the sample increases. And the Low gold-embedded MBs suspension demonstrated the biggest increase in contrast of about 20 times.

Table 2. Contrast of Gold nanoparticles, Plain MBs suspension, and Low gold-embedded MBs suspension

Sample name Contrast with voltage of 55kV Contrast with voltage of (%) 90kV (%) Gold nanoparticles 1.59 4.07 Plain MBs 12.34 20.70 Low gold-embedded 0.16 3.68 MBs

In the second group of experiments, water, plain MBs suspension with concentration of 107, 5*106, 106, ml-1, and sedimentation of high gold-embedded MBs were loaded on a microplate. The microplate was placed on the detector. X-rays with 600uA current, and a series of voltages from 30kV to 90kV with step 5kV were applied to the test. Images with exposure time 1000ms were acquired. The exposure time was longer than the previous experiment. Therefore, more photons were captured by the sensors, which reduces random errors.

The results of the experiments were processed in the same way as the previous experiments.

When the voltage went higher than 55kV the detectors were saturated and images were overexposed. Therefore, the driving voltage of 55 kV was considered as an upper limit without the filter in our study. 14

Figure 15 is an image of test samples acquired by CT in the second group of experiments.

Figure 15. An image of test samples with X-ray of voltage 30kV, current 600uA, and exposure time 1000ms.

The results of the second group of experiments are shown in Figure 16. The sedimentation of high gold-embedded MBs has the highest contrast. The plain MBs suspension with concentration of 106ml-1 has the lowest, negative contrast. With the increase of the X-ray source voltage, the contrasts of plain MBs suspension drops. However, the contrast of sedimentation increases slightly. When the X-ray source voltage is 50kV, the results for contrast values are unexpected.

40 MBs with concertion of 10^6 30

20 water

10 MBs with concertion of 5*10^6

Contrast(%) 0 MBs with concertion of 10^7 30 35 40 45 50 -10 sedimentation of high gold- -20 embedded MBs Voltage of the X-ray source (kV)

Figure 16. Contrasts of the various concentration of plain MBs suspension and sedimentation of high gold-embedded MBs in

respect to the voltage of the X-ray source without filter.

Discussion

Microscopy test

In the microscopy test, both the mean diameter and the concentration are underestimated, because the microscope focuses only on a very thin plane. This is illustrated in the Figure 17. If the microscope focuses on the plane, the diameter of MB1 is accurately observed, however the diameter of MB2 is underestimated. On the other hand, if the microscope focuses on plane B, the situation is the opposite. Thus no matter where the microscopy focuses, the mean diameter of the MBs is underestimated.

Figure 17. A simplified situation of the microscopy observation

In aspect of concentration, since not all MBs are at the same plane, we cannot observe all MBs. Therefore, the concentration is underestimated.

To get a more accurate result of the diameter and concentration of the MBs, we propose to use the following three methods: 16

 Place the sample on the plate of the microscope and wait for 10 mins. Since the MBs contain the air, it will float to the top. Then most of the MBs are in the close planes. Therefore, the underestimation will reduce. In our experiment, most of the MBs float to the close planes after 10 min.  3D microscopy scanning provides the 3D image of the MBs. The volume of the MBs can be acquired from the 3D image. Since the MBs are approximately spheres, the volume of the MBs can be used to calculate the diameter of the MBs. As a result, the underestimation of the diameter is eliminated. Because the depth of scanning area can be changed, all the MBs can be detected theoretically.  The diameter of MBs is from 3 to 4um. It is close to the cell. Therefore, Coulter counter might be used for characterize size and concentration of MBs.

The result of the microscopy test shows that no high gold-embedded MBs were formed and the low gold-embedded MBs suspension has a much lower concentration than plain MBs suspension. Therefore, we suspect there are some chemical compounds in the suspension of the ExiTronTM nano 12000 which react with the PVA solution and lead to formation of the porous MBs filled with water instead of air. In the future, if we want to fabricate MBs with high number gold nanoparticles, we might consider to conjugate gold nanoparticles to the surface of the MBs.

Ultrasound test

In the ultrasound calibration, the best way to find out the PNP is to perform 3D scanning for the pressure filed. However, 3D scanning with high special resolution is time consuming. In this project, we used two ways to detect PNP.

 Scan the horizontal plane, which is 80 cm (close to the near field) away from the transducer.  Scan the vertical plane in the middle of the transducer. Find the point with highest negative pressure. Then scan the horizontal plane, which contains that point. Record the highest negative in that horizontal plane as PNP.

The results of two methods are close. Nevertheless, the result of the first method is more linear and the first method requires less time. 17

0.35 0.3 0.25 0.2 0.15

PNP(MPa) 0.1 0.05 0 0 5 10 15 20 25 Attenuation (dB)

Figure 18. Relation between attenuation and PNP. The orange points are detected by 1st method. The blue points are

detected by 2rd method.

The reason why results of the 2rd method are less linear than the 1st one is that the pressure field and scanning plane are not perfectly aligned. Figure 19 demonstrates this misalignment. Although all available methods had been used to set the ultrasound beam travel vertically, the vertically 2D scan shows that the beam travels not vertically.

Figure 19. The vertically 2D scan of the pressure beam.

The results of the power spectra measurements show that when the PNP of the pressure is low, the amplitude of the second harmonic is much lower than the fundamental frequency. Therefore, at a low PNP range, MBs should have linear behaviors. However, at high PNP range, nonlinear behaviors may appear.

In the ultrasound test the results show that the MBs with the concertation of 107 and 5*106 ml-1 provide a considerable attenuation. The MBs with a concentration of 106 have a very low attenuation.

MBs with the concertation of 107, 5*106, and 106ml-1 were chosen to perform ultrasound test based on the experience from a previous study of Sciallero et al. [5]

At low pressure range below 100 kPa, the attenuation coefficients from MBs suspension diluted to the concentration of 107 and 5*106 ml-1 are constant and equal 14 dB and 7 dB/cm at a fundamental frequency of 3.5 MHz. However, attenuation at second and third harmonic increases slightly when the PNP goes from 50 to 100 kPa following the increase of excitation pressure (see calibration curves for 2nd and 3rd harmonic). As a result, below 100 kPa, MBs oscillate in a linear fashion.

Above 100 kPa, attenuation coefficient at the fundamental frequency drops but on the second and third harmonic increases indicating the relocation of acoustic energy from fundamental to the higher order harmonics. Thus MBs oscillate in a nonlinear fashion.

When the PNP increases above 200 kPa, the nonlinear behaviors become even more pronounced. The third harmonic starts building up, whereas the attenuation coefficient decreases both at fundamental and second harmonic.

The temperature is an important impact factor. We try to perform the experiments as quick as possible, thus the temperature of water will be almost similar during the test.

CT test

In the first group of CT tests, the plain MBs suspension has the highest contrast; we believe that is because the plain MBs suspension has a very high concentration (about 108 ml-1 level). And results show that when the voltage of the X-ray source increases, the contrast of gold nanoparticles increases about 3 times and the contrast of Low gold-embedded MBs suspension increases about 21 times, while the contrast of plain MBs suspension increases about 1.7 times. Therefore, we suspect that there might be a synergistic effect of absorption high energy (55keV – 90keV) X-ray photons when the PVA is combined with gold nanoparticles.

In the second group of CT tests, at the voltage of 50kV, the light field is over exposured. Therefore, the absolute value of the calculated contrasts of samples (except water) is higher than the real contrast.

The result of the second group of CT test shows that the plain MBs suspension with concentration 106 has a negative contrast. We repeated the experiments and increased the current to reduce the random errors (shown in Figure 20). We confirmed that MBs suspension with concentration 106 has a negative contrast. Therefore, we might find plain MBs suspension with a certain concentration above 106ml-1 provide both considerable negative contrast in CT and considerable enhancement of the backscattered signal in ultrasound imaging. The unexpected results at voltage 45 kV of the X-ray source are caused by the over exposure of the light field. 19

8 6 4 water 2 0 MBs with concertion of 10^6 -2 30 35 40 45 -4 MBs with concertion of Contrast(%) -6 5*10^6 -8 MBs with concertion of 10^7 -10 -12 Voltage of the X-ray source (kV)

Figure 20. The contrasts of the various concentration of plain MBs concentration respect to the output peak voltage of the CT

with higher current.

The limitation of the current study is that water is considered as reference surrounding media but not the soft tissue in the CT test. If we divide the contrast reference to water and tissue of the same

푐표푛푡푟푎푠푡 푡표 푤푎푡푒푟 휇푠푢푏푠푡푎푛푐푒−휇푤푎푡푒푟 휇푡푖푠푠푢푒 sample, we get the contrast ratio = ∗ , where 휇푠푢푏푠푡푎푛푐푒 is 푐표푛푡푟푎푠푡 푡표 푡푖푠푠푢푒 휇푠푢푏푠푡푎푛푐푒−휇푡푖푠푠푢푒 휇푤푎푡푒푟 the average linear attenuation coefficient of substance contain in the sample, 휇푤푎푡푒푟 is the average linear attenuation coefficient of water, and 휇푡푖푠푠푢푒 is the average linear attenuation coefficient of tissue. Since the tissue has a higher average linear attenuation coefficient than water. By simple math, we can prove that the contrast of a sample reference to water is higher than the contrast reference to the tissue, in case that the contrast is positive. On the other hand, if the contrast of the sample is negative, the change of the contrast is uncertain. However, the difference between the contrast reference to water and contrast reference to tissue should be small (unless the 휇푠푢푏푠푡푎푛푐푒is very close to the 휇푤푎푡푒푟), because the 휇푡푖푠푠푢푒is quite close to the 휇푤푎푡푒푟.

The reason for tissue having a higher average linear attenuation than water is that the tissue has a higher mass attenuation coefficient for photon energy 1keV to 8keV than the water, and a similar mass attenuation coefficient for photon energy 8keV to 55keV as the water [6,7].

Another limitation of the CT test is that the X-ray beam does not travel vertically. Therefore, the density of the photons is higher in the center of the detector and lower in the corner (shown in Figure 21). To minimize the effect of this limitation, we tried to put the center of all samples in the center of detector, making all samples equal.

X-ray source

X-ray beam

Detector

Figure 21. Simplified model of the CT. The arrows present the X-ray beam. The X-ray beam has a higher density X-ray beam in the center. 21

Conclusion

In the work presented here, we found that sedimentation of high gold-embedded MBs has a considerable contrast in CT. Therefore, if we could fabricate stable high gold-embedded MBs suspension with concentrations, it may be a promising hybrid contrast agent.

The plain MBs suspension with concentration 106 has a negative contrast. Therefore, plain MBs suspension with a certain concentration above 106ml-1 demonstrating both considerable negative contrast in CT and enhancement of the backscattered signal in ultrasound imaging might be found.

There may be a synergistic effect of absorption high energy (55keV – 90keV) X-ray photons, when the PVA is combined with gold nanoparticles.

Further work need to be done to access the feasibility to fabricate high gold-embedded MBs suspension with concentrations higher than 5*106. CT tests for various concentrations of plain MBs suspension with small concentration step need to be done to find out whether it is possible for the plain MBs suspension to provide both considerable negative contrast in CT and considerable enhancement of the backscattered signal in ultrasound imaging.

Acknowledgment

I want use this chance to acknowledge everyone who helped me throughout my master thesis project.

First of all, I would like to thank my supervisor Dr. Dmitry Grishenkov who provided me timely guidance. Without his help, I would not have finished my thesis.

I am also thankful to Professor Birgitta Janerot Sjöberg, who gave me many comments on my report.

The helpful discussion with my group supervisor Dr. Chunliang Wang is greatly appreciated

Many thanks are due to Professor Massimiliano Colarieti Tosti, doctoral students David Larsson, and Philip Sundqvist. They helped me to perform the CT test and explained many questions regarding matlab and CT.

A special thanks to the China Scholarship Council for funding this project.

I highly appreciated the help of Li Zehang with 3D model building.

Finally, I would like to thank my friends Xueying Zhong, Julian Conrad, Ling Mingzhu, and my family for their support.

References

[1] Kim, D., Yu, M. K., Lee, T. S., Park, J. J., Jeong, Y. Y., & Jon, S. (2011). Amphiphilic polymer-coated hybrid nanoparticles as CT/MRI dual contrast agents. Nanotechnology, 22(15), 155101. [2] Brismar, T. B., Grishenkov, D., Gustafsson, B., Härmark, J., Barrefelt, Å., Kothapalli, S. V., ... & Paradossi, G. (2012). Magnetite nanoparticles can be coupled to microbubbles to support multimodal imaging. Biomacromolecules, 13(5), 1390-1399. [3] Zheng, M. (2011). Ultrasound contrast agents: fabrication, size distribution and visualization (Master of science thesis). KTH [4] Nebuloni, L., Kuhn, G. A., & Müller, R. (2013). A comparative analysis of water-soluble and blood-pool contrast agents for in vivo vascular imaging with micro-CT. Academic radiology, 20(10), 1247-1255. [5] Sciallero, C., Grishenkov, D., Kothapalli, S. V., Oddo, L., & Trucco, A. (2013). Acoustic characterization and contrast imaging of microbubbles encapsulated by polymeric shells coated or filled with magnetic nanoparticles. The Journal of the Acoustical Society of America, 134(5), 3918-3930. [6] X-ray Mass absorption coefficient of water. In The National Institute of Standards and Technology. Retrieved May 27th, 2016, from http://physics.nist.gov/PhysRefData/XrayMassCoef/ComTab/water.html [7] X-ray Mass absorption coefficient of tissue soft. In The National Institute of Standards and Technology. Retrieved May 27th, 2016, from http://physics.nist.gov/PhysRefData/XrayMassCoef/ComTab/tissue.html

Appendix

Introduction

Contrast agents improve the diagnostic values of various imaging modalities [1]. For instance, in echocardiography contrast agents are used in percutaneous coronary intervention to increase the visibility of vessels [2].

There are different types of contrast agents currently used in clinic but also under research investigation. For example, the microbubbles (MBs) filled with gas and Iodine are clinically used as contrast agents in the ultrasonography and computer tomography (CT), respectively. Gold nanoparticles is an under research contrast agent for CT used in preclinical test and translational research.

Recently researchers try to combine different imaging modalities to form new hybrid imaging modalities. Typically, this new hybrid imaging modality maintains and combines the advantages of the parental modalities and in general is more powerful than the sum of its parts [3]. For instance CT/ ultrasound imaging modality helps to perform tasks which require both high spatial and time resolution. Prof. Jose Zamorano [4] used CT/ ultrasound imaging modality to find the optimal location to place the pace maker lead in the left ventricle. Doing CT and ultrasound imaging alone can’t localize the optimal placing place. However, when they are combined, they create synergistic effects. More information is gained than the sum of the information they provide alone. Similar synergistic effects happen when other imaging modalities are combined.

To improve the diagnostic outcome in hybrid imaging modalities, multimodal contrast agents need to be developed. Hybrid contrast agents for CT and ultrasound (CACTUS) is one of those desirable hybrid contrast agents for the modern medical imaging.

Polyvinyl alcohol (PVA) MBs are one of the latest generations of ultrasound contrast agents (UCAs). Previous studies [5,6] shows that the PVA shell is able to incorporate pharmacological material chemically or mechanically, such as iron oxide and nitric oxide.

Therefore, two methods for developing hybrid contrast agents were proposed. First method is to combine MBs with gold nanoparticles which is an under research contrast agent for CT. Second method is to determine at what concentration plain MBs have both considerable negative contrast in CT and enhancement of the backscattered signal in ultrasound.

Imaging technique

Ultrasound

Ultrasounds are pressure waves with frequencies higher than the audible range.

Medical ultrasound application

Ultrasound can be used as a diagnostic imaging technique in the medical area. It helps to observe the growth of the fetus, the movements of the heart ventricles and valves etc.

The ultrasound imaging is widely used in the clinic. Compared with other imaging modalities, ultrasound imaging has those advantages:

 It is cheaper than other medical imaging modalities.  It has no ionizing radiation.  Long-term side effects is reported，in case that ultrasound imaging is used according to recommendation.  It has the highest temporal resolution among all the imaging modalities.  High frequency ultrasound imaging has a high spatial resolution.  Portable ultrasound imaging devices are commercial available.

However, the ultrasound imaging is highly operator-dependent. Therefore, the experience of operators has a great impact on the quality of images and accuracy of diagnoses [7].

Attenuation, reflection and scattering of ultrasound

When the ultrasound waves propagate though media with viscosity, the energy of the ultrasound waves is gradually lost over the distance travelled. This effect is known as attenuation [8].

When the ultrasound waves propagate from one media to another media with different acoustic impedance to the previous media, some energy of the ultrasound waves is reflected back at the boundary of two medias. This effect is known as reflection [8].

However, if the second media is a very small target comparable to the wavelength of the ultrasound waves. The reflection no longer follows the law of reflection for the large interface. Instead, the waves scatter in many different angles. This effect is known as scattering [8].

The reflected echoes and the scattered echoes form the ultrasound images [9]. The different reflected echoes and scattered echoes from different tissue results in different the brightness in the 26

images. By enhancing echoes from a specific tissue e.g. blood, we can increase the visibility of that tissue. On the other hand, the attenuation decreases the energy of the ultrasound waves. Therefore, tissue that is located distal to the contrast enhanced region (cause significant attenuation to the waves) will appear less echogenic and thus darker on the image.

Main parameters of ultrasound wave

Peak negative pressure (PNP) is the minimum value of pressure in the medium during the ultrasound pulse. High PNP might cause damage to tissue.

Mechanical index (MI) is an attempt to estimate the degree of cavitation related bioeffect. High MI might cause serious consequence to the tissue.

Intensity represents how much energy per area passing through at the measurement point [8].

Pulse average intensity is mean intensity of ultrasound wave only during the pulse duration.

Temporal average intensity is mean intensity of ultrasound wave during the pulse repetition period.

X-ray

In 1895, Wilhelm Conrad Röntgen discovered X-ray. It was used in the clinic for the first time in 1896. Since then X-rays had become one of the most important discoveries in the clinic area.

Production of X-ray

The production of X-ray in the X-ray tubes follows almost same procedure. First the electrons are released from the heated filament and accelerated to strike the metal target. In result photons are released and form X-ray beam [10]. The photons have different energies and the peak energy of the photons is lower than the energy of incidence electrons.

X-ray can be roughly classified into two catalogues by wavelength [11]

 Hard X-ray has a wavelength range of 0.01-0.1 nm.

 Soft X-ray has a wavelength range of 0.1-10nm.

The hard X-ray is used in the medical imaging, because hard X-ray is able to traverse relatively thicker objects than the soft X-ray.

X-ray absorption

X-ray experiences 3 types of interactions in matters [12].

 Photoelectric effect  Scattering  Pair production

As a result of the interactions, the photons of the X-ray lose part of their energy or are completely absorbed. Therefore, the photon intensity will decrease. The decrease of the intensity is described by the Beer law [12]:

−휇푥 I(x) = 퐼표푒 (1) where μ is the linear attenuation coefficient and the x is the thickness of the sample.

I(x) is the radiant flux transmitted. I0 is the radiant flux arrive at the sample.

Medical application

There are many medical applications based on X-ray.

Radiographs are 2D X-ray images. During the test, X-ray traverses through the patient’s body. The intensity of X-ray reaches the detector demonstrates what kind of the tissue this X-ray has traversed through. For example, bones have a high absorption of X-ray, therefore, the intensity of X-ray, which traversed through bone reduce more in its shadow. Due to this working principle. Radiographs play important roles in the diagnosis and monitoring of pathology of the bones. Since only a short X-ray pulse was used during the imaging. Therefore, patients get a very limited exposure [11].

Computed Tomography is a 3D diagnostic imaging modality. The 3D images of CT are reconstructed from the X-ray projections of the scanned object obtained at different angles. Interested cross-sections are visualized at certain planes [13]. Compared with the 2D X-ray, CT provides much more information. Depending on the diagnostic demands, the 3D image can be observed in different planes. Moreover CT enables users to distinguish structures with extremely small differences in Hounsfield units. However, patients receive more radiation doses in the CT than a 2D X-ray, which potential risk to the patients.

Because of its own characters, CT is commonly used to detect cerebral infarction, tumors, to observe the anatomical structure changes, etc.

Contrast agents

Contrast agents are substance used to improve the visibility between structures of interest and their surrounding environment.

There are different kinds of contrast agents:

X-ray contrast agents

X-ray contrast agents are medical contrast agents, which work with X-ray-based imaging to improve their visibility of internal structures [14].

Absorption coefficient. The absorption coefficient is the attenuation in intensity of the incident X-ray per unit distant of the material [15]. The high atomic number materials usually have a higher absorption coefficient. The absorption coefficient curves of those materials are not smooth. For example, there is a sharp increase of absorption coefficient curve of gold (shown in Figure 1) near the photon energy of 80.7 keV. This sudden increase at photon energy of K-edge is because of the photoelectric absorption of the K shell [16].

80.7 keV K edge

Figure 1. X-rays mass absorption coefficient of gold (Au) respect to the photon energy [17]

Ultrasound contrast agents

UCAs are substances, administered in the blood pool or in a cavity, used to increase ultrasound echo [18,19].

Development of UCAs. UCAs improved the accuracy and reliability of ultrasound imaging in the past decades [19,20].

At the beginning the UCAs are simply free air MBs. However, those MBs have a very short lifespan. Later the stability of MBs was improved by coating galactose, albumin, or polymers [19]. Those shells increase the lifespan of the UCAs. Following this, Low solubility gases, such as perfluorocarbons, were introduced to dramatically improve persistence of UCAs [21]. Recently, PVA is used to make the shells of UCAs. (shown in Figure 2) The shell thickness of the PVA-shelled MBs reaches 30% of the external radius [22]. Therefore, the PVA-shelled MBs have a better stability and longer lifespan than the previous UCAs. Beside the PVA-shelled MBs are not limited to be used as UCAs, multiple application of PVA-shelled MBs are under developing. For example, Grishenkov et al. trapped nitric oxide inside PVA-shelled MBs to develop a microdevise for theranostic of myocardial ischemia [23].

Figure 2. Structure of the PVA-shelled MBs

Hybrid imaging and contrast agents

Hybrid imaging

Hybrid imaging refers to a new technique, which is formed by the combination of two or more imaging techniques. The new technique usually has the advantage of all the subset imaging techniques. For example, SPECT/CT allows the combination of the exquisite anatomic details from CT with the important functional, physiologic or metabolic information from Single-photon emission computed tomography (SPECT) [24].

The main clinically approved hybrid imaging techniques are listed in Table 1

Table 1. Examples of applications

Combination Advantage Application SPECT/CT  Improve attenuation correction in SPECT.  Primarily endocrine  Overcome the low spatial resolution of the and neuroendocrine radionuclide imaging diseases  Increase accuracy in recognition of the  Myocardial perfusion anatomical structure and area of disease  Infection/inflammation [25]. benign  Malignant skeletal diseases  Radio-guided operation

Positron  Improve accuracy in extent of functional  Detection of unknown emission abnormalities [26] primary tumors [26,29] tomography  Reduce noise in attenuation correction of  Cancer staging and (PET)/CT the PET data [26] therapy monitoring  Improve accuracy of the predicts of PET [27] [26]  Provide more diagnostic value compared to PET or CT separately [28] Magnetic  Provide accurate anatomic information for Neuroimaging is the most Resonance functional abnormalities [30] promising application of Imaging  Compared with PET/CT, MRI/PET provide MRI/PET [30] (MRI)/PET better diagnosis of metastatic lymph nodes in patients with uterine cervical cancer [31].  MR/PET potentially have better diagnostic performance than PET/CT in soft-tissue 32

anatomic information, functional imaging capability and multiplanar image acquisition [32]  Compared with PET/CT, MR/PET has less ionizing radiation

MRI/SPECT  Overcome the lack of anatomical landmarks  Assessment of regional of SPECT cerebral perfusion [33]  Radionuclide therapy of cancer in abdominal and thoracic areas [34]

MRI / CT  Minimize residual errors Potential applications [35]:  Combine anatomical and functional information  Guiding radiation  Reduce ionizing radiation [35] therapy  Analyzing atherosclerotic plaque  Evaluation of acute strokes and brain injuries

Ultrasound/CT  Increase diagnostic confidence [36] Percutaneous  Enhance detectability of small lesion [37] radiofrequency ablation of  Reduce radiation exposure [36] small hepatocellular  Allows precise monitoring of interventional carcinomas [37] procedures [36]

Ultrasound/MRI  Increased diagnostic confidence [36]  Image-guided biopsies  Provide motion correction. [38 39] [38]  Increase the data acquisition windows  Measure the brain shift during free breathing [38] caused by craniotomy  Integrate the high spatial resolution of MRI [39] with the high time resolution of ultrasound Multiple image Close the gaps between different imaging  Under development tech [40] tech

Hybrid contrast agents

Hybrid contrast agents refer to the contrast agents used in the hybrid imaging. Most of the hybrid contrast agents are still under development. Usually the hybrid contrast agents are the combination of multiple contrast agents which are already used. Table 2 reports the list of hybrid contrast agents

Table 2. Examples of Hybrid contrast agents

Imaging tech Component Fusion method CT/MRI Au–Fe3O4 nanoparticles, and Poly(DMA-r-mPEGMA-r-MA) and poly(DMA-r-mPEGMA-r-MA)[41] Au–Fe3O4 nanoparticles solution was sonicated and stirred. Finally the poly(DMA-r-mPEGMA-r-MA) coat around the nanoparticles[41]. Ultrasound/MRI Iron oxide and MBs The magnetic nanoparticles were trapped in the PVA shelled MBs [5,6]. PET/MRI Superparamagnetic iron SPIO was entrapped in a oxide(SPIO), hydrophilic shell, the hydrophilic shell. The radionuclide, and an antibody radionuclide, and an antibody stick to the surface of the hydrophilic shell [42].

Microbubbles platform

PVA-shelled MBs are the latest generation of UCAs. During previous EU FP6 project “S.I.G.H.T.” and FP7 project “3MiCRON” novel polymer MBs that offer increased chemical and mechanical stability were developed [43]. PVA-shelled MBs potentially can be used to load multiple contrast agents to form another hybrid contrast agent. For example, Brismar et al. proposed to use PVA-shelled MBs loaded with iron oxide as hybrid contrast agents for ultrasound/MRI imaging [5].

References

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