Improved MRI Cancer Imaging Using Gadolinium as the Contrast Agent in Short

Single Walled Carbon Nanotubes

Dr. Mary Frame McMahon

Group CJuan Bastidas

Eric D’AmbrosioMohammad Reda El Mkhantar

James MutinoPeter NazaroffJaekang Yoo

Faculty Advisor Dr. Balaji SitharamanUndergraduate TA Samantha Rossano

Background

Cancer is a disease that has been found in humans and other animals

since the beginning and has become recently more infamous. According to the

American Cancer Society website over 13 million people in the United States

have had some sort of invasive cancer, and that one in three Americans will have

some sort of cancer in their lifetime[1].

Cancer is a disease caused by unregulated cell growth of abnormal cells.

Cells become cancer cells because of some sort of DNA damage. DNA damage

can come from a wide range of places; mutations can be genetically inherited,

caused by some malfunction in cellular mitosis or caused by external forces such

as radiation[1]. There are even viruses known as Oncoviruses, which can cause

cancer[1].

Cells become cancerous when they continue to grow and proliferate when

they shouldn’t be. In eukaryotic cells, cell division occurs by two processes,

mitosis and meiosis. Meiosis is only used to create gametes for sexual

reproduction. Mitosis occurs in humans for reasons such as growth and repair

and is usually regulated by certain checkpoints. The first checkpoint is known as

the G1 checkpoint or the restriction checkpoint[6]. This checkpoint is where the

cell decides whether it should divide, delay division or enter a resting phase.

Their surrounding cells, or environmental conditions signal healthy human cells

when it is time to enter mitosis [6]. Cancerous cells proliferate no matter what

kind of external signaling they receive[1].

Cancer cells can sometimes form into clumps that are known as tumors.

Many methods for detecting cancer involve the imaging of tumors, however some

cancers such as leukemia (cancer of the blood cells) do not form tumors and

some tumors aren’t cancerous [1]. Tumors that are benign can grow and press

up against other organs, but will not spread. The spreading of cancerous cells

from their point of origin to other organs is known as metastasis[6]. Tumors that

are capable of metastasis are known as malignant tumors[1].

The first recorded description of cancer was in Egypt around 3000 B.C.

and refers to 8 cases of tumors on the breast [1]. The origin of the word Cancer

comes from the Greek “carcinos” and “carcinomas”, which Hippocrates used to

describe tumors that did or did not cause ulcers[1]. Like most other diseases, the

lack of technology severely limited our early understanding of cancer. The

development of certain enabling technologies and improvements in cancer

imaging has improved our understanding of this disease dramatically.

            Until the 19th century and the development of the modern microscope,

most information from cancer came from autopsies. During the 1700s John

Hunter, the Scottish surgeon, suggested that certain cancers could be

removed[2]. However without any sort of imaging technology a surgeon would be

forced to go into surgery blind and try to find the tumor and hopefully it could be

removed. 

With the invention of the compound light microscope and the work of

Rudolf Virchow the study of cancer pathology was born. Virchow used

microscopes to study cancerous tissues at the microscopic level[2]. His work

allowed him to study the damage that cancer did to tissues, and also allowed the

study of tumors once they had been removed to obtain a better diagnosis.

The 19th century also saw an increase in cancer imaging technology.

Inventions such as the gastroscope and the cystoscope were invented in the late

1800s and used to detect cancer in the lower esophagus/stomach and bladder,

respectively [2]. In 1896 Dr. Franz Konig used the newly invented x-ray to

examine a leg that he had amputated and discovered that there was a sarcoma

of the tibia [2]. The invention of the X-ray and the discovery that it can be used to

find tumors inside of the body started the modern age of cancer imaging and

paved the way for more advanced imaging techniques[2].

During the twentieth century we observed the use of our most advanced

technologies in the field of cancer imaging and the invention of techniques that

we use today. In 1902 Willem Einthoven took the first ECG reading; ECG’s are

used to diagnose renal cancer [2]. In 1941 George Papanicolaou invented the

technique known as the Pap smear for detecting pre-cancerous and cancerous

cells inside the female genital tract [2]. In 1951 Raul Leborgne used a cone and

compression pad to X-ray human breasts in order to image breast cancer, and

paved the way for the modern mammogram [2].

The 1970s saw monumental improvement in cancer imaging and gave

birth two three of our modern forms of noninvasive medical imaging. In 1972

Godfrey Hounsfield used X-ray imaging and computer assisted analysis to create

cross sections of organs and other body parts[2]. This invention is known as the

CT scan.

In 1974 the first positron emission topography scanner was used[2]. PET

scanners use the detection of radiation from chemicals introduced to the body to

produce a high-resolution computerized image, which show biochemical activity

of observed structures. In 1973 one of the most important inventions in modern

cancer imaging came into existence[2]. Magnetic resonance imaging reads a

radio frequency emitted by excited hydrogen atoms in the body as the result of a

large and uniform magnetic field[13].

Before MRI, we used x-ray to investigate human body [6]. There are two

ways to use X-ray, conventional x-ray imaging and computer tomography [6].

To begin with, x-ray is a form of electromagnetic radiation. It can pass

through solid objects so that it can be used to create images of human body, in

which the image is a spatial map of the object’s susceptibility to penetration by

the rays [6]. X-rays have two useful properties for imaging [6]. First, X-rays

penetrate the human body translucently at certain wavelengths [6]. It means the

rays pass through the body, also, they are partially absorbed as they penetrate at

the same time [6]. The denser tissue density there is in the path of the x-rays, the

more the fraction of radiation that is absorbed [6]. Second, x-rays have the ability

to expose photographic film like visible light [6].

For conventional x-ray imaging, the x-ray beam penetrated through a

body and will expose film [6]. An x-ray image is a negative image [6]. The film is

darker where the tissue of the body is less dense and lighter where is more

dense, which means dark parts occur where the body has lighter elements such

as flesh, allowing more x-rays to penetrate through [6]. In contrast, light regions

occur where the body is dense with heavier elements such a bones, which allows

fewer x-rays to expose the film [6]. In other words, an x-ray image is like a

shadow [6]. The conventional x-ray process can just make two-dimensional

image with three-dimensional body [6]. It just represents the penetration of

radiation through the body onto a two-dimensional plane [6]. This kind of image is

called a projection [6]. X-ray images can be used in medical inspection because

it can show fractures and breaks in bones, fluid in lungs, cavities in teeth and

cancer in breasts [6]. If there is a big difference in the density of the tissue, such

as with soft tissue and air, bone and soft tissue, or water and soft tissue, it can

provide good contrast and make nice images [6]. The most common applications

of x-ray imaging is the chest x-ray, which physicians use to search infection

within the lungs, fractures in the bones of the rib cage, and certain kinds of heart

disease [6].

However, CT uses x-rays in a different way. It uses x-ray to

produce images of specific areas of the scanned object, which allows the user to

see what is inside it without cutting it open [6]. When the patient goes into the CT

imaging system, rotating x-ray sources work within the circular opening, and x-

ray detectors also rotates in synchrony all around the patient [7]. The x-ray

source makes a narrow, fan-shaped beam [7]. The patient is moved into x-ray

generator and the detectors and it can create an image of one cross-section

through the body at a time [6]. The table moves the patient’s position to image

each slice [6]. Computer will process all the data to prepare a series of image

slices into a three-dimensional view of the certain organ or body region [7].

However, since CT uses x-rays to make images, there can be a risk of damage

to DNA in human body, which can cause cancer [7]. In comparison, MRI does

not use x-rays and there is less chance to expose to damage [6].

MRI, which is used as device in this paper, is a medical imaging

technique by visualizing internal structures of the body [6]. It provides detailed

three-dimensional images, especially of soft tissue that cannot easily be imaged

in other modalities, such as CT [6]. MRI is also pretty versatile. We can use it in

many different ways such biochemical composition, tissue function, and

molecular diffusion, as well as structure [6]. MR scans properties of the magnetic

dipole (spin) of atomic nuclei at magnetic fields [6]. It uses hydrogen nuclei

(protons), which will be aligned in a large magnetic field [6]. When a person is

placed into the magnetic field of an MR scanner, the magnetic moment vectors of

their hydrogen nuclei align parallel with the direction of the field [6]. A short radio

frequency of electromagnetic radiation is applied in a plane perpendicular to

magnetic field, creating a new magnetic field, which is called transverse magnetic

field [6]. This radio frequency is known as the resonance frequency that flips the

spin of the protons in the magnetic field [6]. After the electromagnetic field is off,

the hydrogen nuclei spontaneously begin to return to their original equilibrium

configuration [6]. During this process, they release RF energy, which can be

found by the receiver coils surrounding the person [6]. The produced signals are

recorded and the resulting data are processed to generate an image [6].

Hydrogen nuclei in different parts return to their original equilibrium configuration

at different relaxation rates [6]. The returning time to equilibrium position is

categorized by the longitudinal relaxation time (T1) and the transverse relaxation

time (T2) [6]. The amount of brightness in MR is resulted by proton density, T1

relaxation, and T2 relaxation within the particular tissue [6]. T1 relaxation shows

nice contrast for different types of soft tissue, while T2 relaxation is good for

pathology [6].

MRI imaging has improved since 1973 with the advancement of

computers and the technology surrounding the MRI [2]. However the MRI

scanner is still limited in resolution by the T1 relaxation times of hydrogen atoms,

which is about 3600 ms [33]. Finding small masses of tissue that don’t belong

can be difficult with this limited resolution [33]. In order to identify tumors in a

more efficient way there needs to be better contrast in MRI imaging[33].

Thanks to the nature of the MRI machine, there are certain chemicals and

chemical compounds that improve the contrast of MRI images. The fact that MRI

becomes common in use of medical purpose has made the invention of a new

kind of pharmacological products, which is called contrast agents [8]. MRI

contrast agents are injected to enhance the image of blood vessels, tumors or

inflammation [8]. By far the most common is the gadolinium-based agents [8].

Gadolinium is a high paramagnetic ion with seven unpaired electrons and has

comparably long electronic relaxation time, which makes it an excellent

relaxation agent [8]. It disturbs the local magnetic field of nearby protons and

results in a shortening T1 and T2 relaxation time [8]. It means relaxation rate

increases, either longitudinal (1/T1) or transverse (1/T2) [8].

These MRI contrast agents have a shorter T1 time then hydrogen atoms

and help MRI images produce a more detailed image of the area in question[8].

Most MRI contrast agents are derivatives of the Gadolinium^3+ ion[4].

Gadolinium is a silvery white metal that is malleable and ductile [3]. What

makes Gadolinium important as a contrast agent is it’s symmetry, magnetism

and the fact that it has the highest number of unpaired electron spins (7 unpaired

electrons) [3] , which gives Gd(III) at 5 ppm a T1 time of 1600 ms [33]. This

shortened T1 relaxation time produces a clearer image in an MRI and makes

spotting things like tumors much easier[5].

Gadolinium is a very useful tool in cancer imaging, but unfortunately there

are some problems that can arise from its use. Gadolinium has been known to

cause a rare but serious disease in some people who have kidney problems [4].

This disease is called Nephrogenic Systematic Fibrosis (NSF) and causes

fibrosis in the skin and connective tissue and can even result in death [4]. If there

was a way that Gd(III) could be in the body so that it could improve MRI contrast

but be encapsulated so that it does not react with any parts of the body then this

problem could potentially be eliminated[4].

Ultra short carbon nanotubes allow for gadolinium to be present in the

area of the body being imaged, without exposing the ion directly to anything else

in the body. Carbon nanotubes are a cylinder-shaped nano-material, which is

made of carbon [9]. They have a lot of kinds of structures, length, thickness, and

number of layers [9]. Carbon nanotubes usually have up to 50 nm diameters [10].

Their lengths are typically several microns, but recently they can be made much

longer, and measured in centimeters [10]. Carbon nanotube was highlighted from

when we could research and deal with carbon tubules in nano-meter dimensions

[9].

Carbon nanotubes are usually produced by three methods: arc-

discharge, laser ablation, and chemical vapor deposition [10]. Among three

methods, the last one is the most widely used commercial method to prepare

carbon nanotubes [10]. This process generally involves reaction of a metal

catalyst with a hydrocarbon at very high temperatures to produce carbon

nanotube [10]. Nanotubes, which are made by chemical vapor deposition,

commonly have metal catalysts on the surface of outside of the nanotube [10].

Because metal catalysts, typically nickel can be used to make carbon nanotubes

bigger, there can be a problem with carbon nanotubes being cytotoxic [10].

Therefore a purification step is highly required before we can use carbon

nanotubes for biomedical applications [10].

There are also several methods for purifying carbon nanotubes [10]. The

most popular method is refluxing carbon nanotubes in an oxidizing acid such as

nitric acid [10]. This process includes oxidizing and removing the metal catalysts

from both the inside and outside of the tube [10]. Besides, any defects in the tube

can also be oxidized while it makes additional groups of carboxylic acids along

the tube [10]. These carboxylic acid groups can be more functionalized allowing

tuning of the surface chemistry of the nanotube [10].

Carbon nanotubes become popular for biomedical applications because

of its composition, high aspect ratio and properties [10]. The number of articles

about it has been doubling each year since the year 2000 [10].

Design Criteria

In the invention of Shortened Carbon Nanotubes in [21] by Wilson and

Bolskar, they illustrate a carbon nanotube of a distance end to end in range from

20 nm to 50 nm [21], and containing gadolinium as a contrast agent [21], which is

able to significantly improve the level of detail in the MRI, therefore increasing its

image quality [21]. In fact, we want to expand the relaxivity [11], known as the

variation of the proton’s relaxation rate divided by its particular molarity, and the

standard units are mM^-1*s^-1 [11].

In order for this specific invention [21] to work perfectly, we have several

special conditions in [21] which will further be discussed here.

The design in [21] includes a carbon nanotube, meaning a category of

fullerene, containing an extended cylinder that is composed of 5 and 6

components in each ring [21]. There can be single walled carbon nanotubes,

which include only one cylinder around an axis [21], and there can also be multi-

walled carbon nanotubes, which enclose two or more than two cylinder of

carbons around a specific axis [21]. Moreover, their sizes might vary, and so the

ideal carbon nanotubes for the invention in [21] will be a single walled carbon

nanotube and in the range of 20 nm to 50 nm [21].

There is a big difference between both single walled and multi walled

shortened carbon nanotubes [21]. In this paper the main focus is on single walled

carbon nanotubes [21]. Carbon nanotubes are all composed of folding graphite

layers into carbon cylinders which can form single layers or multiple layers [20].

Single walled carbon nanotubes are the best choice for bio imaging in such

circumstances as an MRI [20]. This is because in general, single walled have a

smaller diameter then multi walled nanotubes [20]. The first big issue with multi

walled carbon nanotubes is that it is more difficult to fill them with contrast agents

[20]. Transport of the contrast agent into the nanotubes is reliant on holes

opening in the tubes at high temperatures [19]. There is little to no open gates

(holes) in multi walled carbon nanotubes since there is not much change in

voltage at the walls [20]. This being said it is much harder for contrast agents

such as gadolinium to permeate the nanotube [20]. The opposite is true for single

walled carbon nanotubes which are much more permeable under the same

conditions [20]. Single walled nanotubes have a much higher conductance at the

walls when heated, which provides a higher carrying density and ability to

transfer through holes in the walls [20]. The second big issue with multi walled

carbon nanotubes is that they will have much more trouble showing up in bio-

imaging then single walled [20]. This lowers resolution in imaging because of the

thicker diameter of the walls, and the fact that there is a low open gate effect so

electrons are not detected as easily from the outside of the nanotube [20].

Actually, multi walled carbon nanotubes throw off the ability to see the contrast

agents within since they have a larger electronic structure themselves [20].

In addition, single walled carbon nanotubes or SWNTs, have special

properties that fit perfectly in several biomedical purposes [11], especially carbon

nanotubes with sizes from 20 nm to 100 nm [11], are excellent when it comes to

its expulsion from the organism [11]. Also, shortened carbon nanotubes could be

preferred for biocompatibility objectives [11], because the outside of the SWNT

can be used to easily add chemical compounds to increase its tendency to

dissolve [11]. Moreover, the inside of the SWNT might be appropriate to place

harmful agents such as gadolinium, and so the toxicity would be enclosed by the

surroundings of the SWNT [11]. These are tremendously important criteria in the

invention [21] by Wilson and Bolskar.

It can be demonstrated that SWNTs having a size from 20 nm to 50 nm,

considerably enlarge the analyzed surface area in [12], and radically reduce the

velocity of discharge of particles [12]. In the study described in [12], short SWNTs

were put side by side with SWNTs that were not divided into the described

length, charcoal and some additional carbon substances that did not illustrate

better characteristics than the ones explained by SWNTs [12]. So because of

these characteristics, the mentioned SWNTs can be implemented in the medical

field to release radionuclide molecules [12]. In addition, the SWNTs were divided

using the practice of fluorination and pyrolysis [12, 21]. In this particular case,

fluorination was done at 100 °C for a time of 2 hours, and at a 1/100 proportion of

F2 in He [12], and to separate the SWNTs, pyrolysis was performed using argon

for 1 hour at 1000 °C [12]. This method is also referred as fluorination-cutting

process, which is also used in the invention in [21] by Wilson and Bolskar.

Once the shortened carbon nanotube has been cut, then we can place a

contrast agent inside the SWNT [21]. This can be done by creating a spread

nanocapsule suspension into water by intense mixing and sonication for 60

minutes at 30 W [21].

In order to understand how the different types of contrast agents behave,

a discussion of the way MRI works is presented. In the MRI, the image quality is

a consequence of the contact between the protons found in the water molecules

and the contrast agents [13]. If a magnetic field is directed through the protons,

their spins become organized in a way perpendicular or in the direction of the

magnetic field [13], and as a result, their spins change to the Larmor frequency

[13]. The process continues by sending a radio-frequency pulse (or RF pulse) as

described in [13], and due to this stimulus, the protons acquire energy and now

change their orientations are in the same direction as this RF pulse [13]. When

the RF pulse is eliminated, protons come back to their initial position [13], and we

can categorize the time taken from the RF pulse to their initial position as T1 or

longitudinal relaxation time, and T2 or transverse relaxation time [13]. This is the

reason contrast agents can also be divided into T1 contrast agents and T2

contrast agents [13, 14], and one of the main tasks of any contrast agent, not

matter if it is a T1 or T2 contrast agents, will be to decrease its relaxation time

[13, 14].

So let us compare T1 and T2 contrast agents. T1 contrast agents are for

the most part paramagnetic molecules [13, 14], and this is because they contain

metals which comprise electrons that are not paired, and as a result, they create

a magnetic moment [14]. Some examples of paramagnetic contrast agents are

manganese, copper, gadolinium and chromium [13 Fig 3(cited in 14)]. On the

other hand, T2 contrast agents include particles in the nanoscale from iron oxide

[13, 14], which are a type of super paramagnetic agents [14].

Although T2 contrast agents from iron oxide have been used significantly

for the past 2 decades [13], they have major difficulties that are preventing them

to desirable contrast agents [13]. These inconveniences include the fact that

these T2 agents are negative imaging agents [13], which means that they

diminish the signal result and resolution, displaying a dim and unclear image that

could be leading to observe diseases in a certain way, where in fact the patient

has a totally different disease [13]. In addition, they have elevated vulnerability,

causing a deformation in the magnetic field of the nearby tissue [13], which also

contributes to make the signal difficult to see and it completely ruins the

surroundings of injury images [13]. On the other hand, T1 contrast agents work in

the opposite way because they are positive imaging agents [13]. This means that

they tend to produce a luminous and resplendent signal that improves the image

quality, leading to the right interpretation of diseases [13]. Moreover, T1 contrast

agents provide a greater spatial resolution, and because they are paramagnetic,

they do not cause the deformation of the constant magnetic properties in

extended magnitudes [13], giving excellent contrast in the surroundings of

anatomic images [13].

Now that we have seen why T1 contrast agents are preferred over T2

contrast agents, let’s examine some of the T1 contrast agents that might be

appropriate for the design in [21]. For example manganese is one of the contrast

agents that can be used in MRI [14, 15]. The total number of unpaired electrons

in Manganese is 5 [15] and it is mainly implemented in animal analysis using

functional MR techniques [15]. An advantage that manganese has is that it

promotes its own stability in the environment [15], but one of its major

disadvantages is the inexistence of compatible molecules that can attach to the

ions of manganese, and the same time, create a stronger adhesion than

molecules derived from manganese itself [15(cited in 14)]. Thus, because

manganese is a poisonous ion [15(cited in 14)], before it can be considered as a

strong contrast agent candidate, it is necessary to discover ways to send it to the

desired location in the body [14], and send it in small amounts, while verifying

that the MRI is able to notice its presence in such amounts [14]. In addition,

manganese has a magnetic moment of 5.92 [13 Fig 3(cited in 14)], but if we

compare it to gadolinium, which has a magnetic moment of 7.94 [13 Fig 3(cited in

14)], it can be concluded that gadolinium provides a somewhat better relaxation

enlargement characteristics than manganese [15]. So based on these facts, we

can recognize that gadolinium is a better candidate to be used as a contrast

agent than manganese.

As stated before, gadolinium is the perfect candidate for the invention in

[21] by Wilson and Bolskar. It is known that gadolinium ion is the best T1 contrast

agent [11], because the number of unpaired electrons that gadolinium encloses

is 7 [11, 13, 21], making gadolinium the only ion with the highest number of

electrons that are not paired [11]. Additionally, gadolinium does not only have an

incredible magnetic moment [11, 13, 21] of 7.94 [13 Fig 3(cited in 14)], but also it

has a relaxing ground state that is not quick, and extremely balanced [11], so it

creates high strength fluctuations close to the Larmor frequency of the protons in

hydrogen, and consequently generating a solid T1 signal [11]. Another

advantage that gadolinium has is that it is very accessible [14].

Furthermore, in [11] and [21], Nuclear Magnetic Relaxation Dispersion or

NMRD is displayed [11 Fig 1, 21 Fig 1]. In this study, the relaxivity of shortened

SWNTs filled with ions of gadolinium, at 37 °C in a solution of 1% sodium

dodecyl benzene sulfate [11, 21] was graphed versus the magnetic field [11, 21].

The concentration for gadolinium was 0.044 mM [11 Fig 1, 21 Table 2]. A

medical contrast agent employed now in MRI is [Gd(DTPA)(H2O)]2- and was also

graphed [11, 21]. It can be seen that the shortened SWNTs containing

Gadolinium ions illustrate a relaxivity equal to 170 mM^-1*s^-1 [11, 21], which is

approximately 40 times bigger [11, 21] than the medical contrast agent which

was 4.0 mM^-1*s^-1 [11, 21]. This was done at a typical power of the MRI field

for the medical visualization purposes from 20 MHz to 60 MHz [11, 21].

The filling of the gadolinium into the shortened carbon nanotubes is very

important [21]. The shortened carbon nanotubes have very high electrical

properties which provide for a good capsule like structure for contrast agents

during MRI’s [19]. Gadolinium is an excellent contrast agent and has a high

resolution inside the SCNT’s during an MRI [21]. The shortened single walled

carbon nanotubes are exposed to dry air at over 400 degrees Celsius [19]. This

hot air opens the ends of the nanotubes [19]. The gadolinium fused with either a

carbon or chloride group is then heated into a vapor and penetrates the nanotube

walls which are open from the heat [19]. The gadolinium then lines up inside the

nanotube and can be confirmed via electron microscopy [19]. The gadolinium

atoms then each transfer 3 electrons to the surrounding area of the SCNT,

creating a steeper electrical resistance vs. temperature curve [19]. This

introduction of the gadolinium into the shortened carbon nanotubes changes the

electrical properties of the nanotubes [19]. Consequently, this creates a much

better contrast during an MRI [19].

In order for carbon nanotubes to be safer they must be biocompatible [21].

It is very important to have biocompatibility with any form of a substance that is

being placed into the human body [6]. For something to be biocompatible it

means that the substance must blend in to the human body and not seem

strange [6]. A substance with little biocompatibility will have negative effects from

the cells in the body [6]. The human immune system will attack any foreign body

substance in order to protect the body [6]. For inserted substances for example,

they stimulate a specific swelling reaction or known as foreign body response [6],

which provokes protein intake, congregation of macrophages and neutrophils,

configuration of giant cells, and at the end, the engagement of endothelial cells

and fibroblasts [6]. However, if the body cannot sense the substance as a foreign

body, then it will not react [6]. This is why it is important to blend in, because

there will not be a negative response if there is high biocompatibility [6].

Biocompatibility is incredibly important when dealing with medical devices and

drugs [6]. The material must assimilate in order to do its job whether it is

something as small as a tiny drug, or as big as a pacemaker [6].

The biocompatibility of carbon nanotubes used for MRI’s must be very

good [21]. The body must allow the single walled nanotubes sealed with

gadolinium (contrast agent) [21] to flow through the body without having a foreign

body response [4]. It is also important that the gadolinium does not leak out of

the nanotubes since it is known to be toxic [4]. The key biocompatibility

components are to make sure the nanotubes are sealed properly, shortened, and

coated in order to mask the fact that they are foreign [21].

It is very important to properly seal carbon nanotubes in order for them to

be effective inside the human body [21]. If the contents of the nanotube were to

leak out inside the body there could be cell and tissue damage [21]. This is why it

is vital that they do not have any holes, and are filled properly [21]. In this case

gadolinium is the contrast agent that is put into the shortened carbon nanotubes

[21]. This metal can be combined with chloride (Cl3) which forms gadolinium tri-

chloride, and makes a very good contrast agent [16]. It is important to have this

combination because the nanotubes must be filled properly and the walls of the

nanotube must be free of defects [16]. The combination of this metal and

chloride nanowires line the inside of the carbon nanotube and decrease the

diameter of the walls [16]. This overall strengthens the walls of the carbon

nanotube and minimizes leakage [16]. There are many different metal nanowires

that that can fill a carbon nanotube, but in this case the focus is on gadolinium

(III) chloride [16]. It is shown that this metal chloride has very defined nanowires

which can be seen through transmission electron microscopy [16].

As previously talked about in the background section of the paper, the

Gadolinium must not get out of the carbon nanotubes [4]. It is a very toxic

substance and does not get along well with the human body [4]. It causes

problem in the kidney, and especially effects people with acute or chronic severe

kidney insufficiency or kidney dysfunction [4]. In some cases it causes NSF

(nephrogenic systemic fibrosis) which causes fibrosis of the skin and connective

tissue [4]. This is just one example of how toxic gadolinium is in the human body.

It is crucial that the SCNT’s are sealed properly and coated so that the human

body does not attack the nanotubes [21]. The goal is to use the gadolinium as a

contrast agent during MRI and then for the body to dispose of it without any harm

[21].

After the sealing process of shortened carbon nanotubes, it is important to

make sure the shortened carbon nanotube will assimilate into the body [21]. The

key factor in making an effective SCNT is to make sure it is very soluble [17].

This is called derivatization, or the coating of the carbon nanotubes to help blend

in [17]. The main way to increasing solubility is with increased bond formation

with carboxylic groups [17]. The big problem is that SCNT’s are very insoluble to

begin with; however they exhibit strong mechanical and electronic properties

[17]. This means that they are open to chemical modifications to the exterior in

particular [17]. SCNT’s are generally wrapped in polymers in order to help

increase the solubility [17]. This increase is very important because once placed

into the body the carbon nanotubes need to perform their imaging task safely and

efficiently [21]. Using mainly 1, 3 dipolar cycloaddition polymers the

functionalization of the nanotubes greatly increased [17]. These polymers create

a very high solubility in water which is perfect for MRI imaging since majority of

the human body is composed of water and is all based in an aqueous

environment [17].

The shortened carbon nanotubes must safely carry the contrast agent and

not harm any cells they come around [4]. Using carbon scaffolding with

carboxylic chains, and phospholipid like structures to the outside of the SCNTs

the biocompatibility inside the human body increases [18]. Through using a wet-

chemical process, defects are created which introduces hydrophilic carboxylic

groups on the ends of the SCNTs [18]. These carboxylic groups react with the

chemicals in the human body [18] without any harmful effects [4]. Oxidized

SCNTs with covalently bonded carbon groups on the walls can also be coupled

with other types of structures such as alcoholic chains to increase solubility [18].

It is shown that by using the addition of carboxylic groups to the walls there is no

high toxicity level [18]. This method shows an efficient way to spread out SCNT’s

into a specific part of the body safely [18]. The carboxylic groups have simple

reactions with those of the human body and then exit soon after [18]. Single

walled shortened carbon nanotubes are great transporters for contrast agents (in

this case gadolinium), within the human body [18]. Consequently, SWCNT’s are

excellent for use in MRI’s based upon their properties, shape and structure [18].

The Design

The invention in [21] by Wilson and Bolskar describes how to make

shortened carbon nanotubes for use in MRI imaging [21]. Robert D Bolskar is a

researcher in the imaging field and a distinguished employee of TDA research

[22]. He has had several publications and many awards won over the course of

his career [22]. For example a well-known publication he co-wrote and won an

award for is the Improved Manufacturing Process for Advanced Imaging Agents

[22]. Lon J. Wilson is a professor of chemistry at Rice University and his research

revolves around incorporating carbon nanotechnology to other fields, namely,

biology and medicine [23]. One of his many publications is Nanotechnology and

MRI contrast enhancement [23].

This invention presents the methods and the advantages of the particular

processes used to make shortened carbon nanotubes [21]. The carbon nanotube

which is a long thin cylinder, for simplification and visualization purposes, will

serve as a net or cradle for the contrast agent that will be inserted inside the

hollow nanotube [21]. So for imaging, the purpose of the shortened carbon

nanotube is to carry magnetic material into the body [21]. A carbon nanotube is a

type of fullerene in the form of a hollow tube consisting of only carbon atoms in

pentagon and hexagon rings [21]. The shortened carbon nanotube has a

diameter of 1.2 nanometer diameter and each carbon ring of the single walled

carbon nanotube has a width of 2.83*10^-10 m, a height of 2.45*10^-10 m and

carbon bond lengths of 1.42*10^-10 m [24]. Also its density is 1.34 g/cm^3 and

its young’s modulus is 648.43 GPa [25]. Carbon nanotubes have lengths in the

order of hundreds of nanometers to a few microns [21]. So the shortened carbon

nanotubes in this invention simply refers to carbon nanotubes of reduced size, in

this case 50 nm would be a perfect size [21].

A long single walled-carbon nanotube of length 100 nm is the starting

material for this invention [21]. First the long carbon nanotube is cut into a

shortened carbon nanotube of about 50 nm by a process called fluorination-

cutting process [21,26]. The long carbon nanotube is reacted with a fluorinating

agent, fluorine [21,26]. This is done by heating the long carbon nanotube in a

fluorine atmosphere at 50°C for 2 hours [21]. The atmosphere is mainly a helium

atmosphere with 1% fluorine [21]. Fluorine atoms from the fluorine gas attach to

the outer or inner surface of the carbon nanotube [21, 26]. CF bonds are formed

in bands around the carbon nanotube [21]. The result is a fluorinated long carbon

nanotube [21, 26]. The following process is pyrolysis which consists of heating

the long fluorinated carbon nanotube at 1000°C for 4 hours in an argon

atmosphere [21, 26]. The heating is done in a quartz tube furnace which is a

temperature programmable heating device [21]. During pyrolysis, the long carbon

nanotubes are cut into short carbon nanotubes along the bands of CF bonds

[21]. CF bonds that did not form in bands but rather in spots in the presence of

fluorine gas, are responsible for the formation of holes in the carbon nanotube as

they volatilize under very high temperature [21]. This formation of holes accounts

for the side wall defects on the shortened carbon nanotube [21]. For purification,

the carbon nanotubes are exposed to high vacuum to remove any still remaining

traces of gases on the carbon nanotube [21]. CF4 and traces of COF2 and CO2

which were generated during pyrolysis are removed [21]. The fluorination cutting

process also involves the removal of residues of yttrium and nickel catalyst

particles that were initially present with the long carbon nanotubes [21]. Y/Ni

(yttrium/nickel) were added as catalysts during the electric arc discharge process

to produce the long carbon nanotubes [21]. The aim here is to have a carbon

nanotube at about 98% free of Y/Ni particles by aqueous acid extraction [21].

This is essential because remaining traces of metal catalysts would interfere with

the magnetic properties of the contrast agent [21].

Following the fluorination cutting process and pyrolysis, gadolinium ion

Gd3+ is inserted as a cargo inside the shortened carbon nanotube [21]. The

loading of the cargo is done by stirring 100 mg of shortened carbon nanotubes

and 100 mg of anhydrous GdCl3 in deionized water at pH 7 which was purified by

high performance liquid chromatography (HPLC) [21]. For better loading,

nanocapsule suspensions are immersed in an ultrasonic bath at 30 W for 60

minutes [21]. The solution obtained after sonication is then centrifuged to collect

the Gd3+ filled short carbon nanotubes which form the pellet [21]. The loaded

short carbon nanotubes form in clumps and the supernatant is then decanted

[21]. The pellet is washed with 25 ml of deionized HPLC grade water and

sonicated in the same manner described previously to remove any traces of

unabsorbed GdCl3 [21]. Again the filled short carbon nanotubes form in the pellet

and the supernatant is decanted [21]. This process is repeated three times [21].

By ICP or inductively coupled plasma analysis, the amount of Gd3+ in the carbon

nanotubes is determined [21]. The mass of the gadolinium loading was

determined to be 2.84% of the total mass of the nanocapsule [21]. The loading of

the contrast agent occurs through the open ends of the short carbon nanotube

but can also happen through the side wall defects [21]. We know that gadolinium

is a toxic substance for the human body [21]. So the presence of side wall

defects can be problematic because if the contrast agent can get in through

those holes then it can also get out [21]. Hence the better carbon nanotube is

one that has the least amount of side wall defects possible [21]. By electron

microscopy, the presence of sidewall defects can be detected [21]. The side wall

defects formation however, cannot be controlled during the fluorination cutting

process [21]. The short carbon nanotubes that have the least amount of holes

will be chosen for further modification [21]. The leakiness of the short carbon

nanotubes is determined by measuring the relaxivity of a solution containing the

filled nanocapsules (filled short carbon nanotubes) using NMR (nuclear magnetic

resonance) [21]. In solution, if free Gd3+ ions leak from the filled short carbon

nanotube then the gadolinium will have a significant effect on the relaxivity of

water protons in the supernatant [21]. If the relaxivity values of the supernatant

decrease after addition of TTHA6- ,then we can confirm that free gadolinium ions

are present in the supernatant because the [GdTTHA]3- complex, which forms

between Gd3+ and the ligand TTHA6-, has a significantly lower relaxivity value

than that of free aqueous Gd3+ ions alone [21].

To measure the relaxivity of the Gd3+ nanocapsules, two solutions are

prepared [21]. The first solution is a saturated solution of 40 mg of the Gd3+ filled

short carbon nanotubes in 20 mL of a 1% SDBS (sodium dodecyl benzene

sulfate) aqueous solution [21]. The second solution contains 10 mg of the Gd3+

filled short carbon nanotubes in 5 mL of a 1% solution of a biologically

compatible pluronic F98 surfactant [21]. After centrifugation, only 10% of the Gd3+

filled short carbon nanotubes, in both solutions, formed a stable suspension [21].

These suspensions present in the supernatant were used for the relaxivity

measurements [21]. The negative controls were Gd3+ filled short carbon

nanotubes suspensions at 40°C and 60 MHz using NMR [21]. The proton

relaxivities of the solutions at 60 MHz and 40°C were measured before and after

the addition of TTHA6- at pH=7 [21]. TTHA6- and Gd3+ form a very stable

complex, [GdTTHA]3- which has no inner-sphere water molecules and thus a

lower relaxitivity compared to aquated Gd3+ ions [21]. The relaxivity

measurements for both solutions however, show that the relaxivities are the

same with and without TTHA6- [21]. This confirms the absence of free Gd3+ ions in

the supernatant, meaning that the gadolinium content does not leak from the

short carbon nanotubes [21]. This is a satisfactory result for this invention since

we do not want the toxic gadolinium to leak from the carbon nanotubes [21]. The

longitudinal relaxation rates (R1) were determined by the inversion recovery

method at pH=7 for the Gd3+ filled shortened carbon nanotubes in the 1% SDBS

surfactant solution and in the 1% pluronic F98 surfactant solution using NMR at

60 MHz [21]. The longitudinal relaxivity (r1) values were obtained from the

equation (T1-1)obs= (T1

-1)d +r1 [Gd3+] in which Tlobs is the relaxation time in

seconds of the Gd3+ filled shortened carbon nanotubes and T1d, the relaxation

time in seconds of the aqueous surfactant solutions [21].

Another test was undertaken to determine the content of the cargo, in

other words, to determine if any traces chemical compounds are present other

than gadolinium ions [21]. First, the 1% SDBS surfactant and the 1% pluronic

F98 surfactant solutions containing Gd3+ filled shortened carbon nanotubes were

treated with a 90% HNO3 (nitric acid) solution and then heated at very high

temperature (specific temperature not mentioned in the invention) until a solid

residue forms [21]. For clarification, all the aqueous solution evaporates [21].

Then, the solid residue is treated with a 30% H2O2 solution and heated again until

all the remaining carbon material, in other words the carbon nanotube structures,

are eliminated [21]. The remaining solid residue is dissolved in 2% HNO3 and

analyzed by ICP [21]. The chemical analysis of this solid residue is done by an

inductively coupled plasma atomic emission spectrometer with a CCD detector

[21]. For each of the two samples from our two solutions, seven scans were done

[21]. In this analysis each chemical is associated to a wavelength [21]. The

gadolinium line was at λ= 376.84 nm [21]. The intensity of the light is associated

with the amount of a chemical [21]. As we can expect the gadolinium line had the

highest intensity because it is the chemical present in the highest concentration

[21]. The line at λ= 361.38 nm indicates the presence of another chemical [21].

Other than gadolinium, Ni or Nickel mentioned earlier was found to be present in

low concentrations from 0.1 to 0.05 ppm (parts per million) as impurity [21]. The

other metal catalyst Y, yttrium was not detected, at least not within the range of

the device which lower limit is 1 ppb (part per billion) [21].

The damaged areas of the tubes have exposed and dangling bonds that are

subject to oxidation by the environment, which cause the bonding of hydroxyl,

carbonyl and carboxyl groups at the end of the nanotubes [27]. These reactions

and bonds of carboxyl groups have been exploited to cause derivatization

(functionalizing) of carbon nanotube and will be discussed further on [27]. These

defects are unwanted and a pristine tube is what is needed to continue on with

the process [21]. After the shortened single walled carbon nanotube has been

examined and deemed acceptable both ends of the tube must be closed [21],

caps are formed and attached to the ends of the tubes with a simple, reliable and

effective technique [28]. Fullerendion opens due to thermal oxidation and causes

hemispherical caps which start single walled carbon nanotube growth at their

open ends [28]. The size and shape of the caps adjusted to the desired

specifications by using specific temperatures of thermal oxidation [28].

After both ends are capped the shortened single walled carbon nanotubes

are derivatized by forming bonds with carboxylic groups to increase solubility in

water, biocompatibility, to lower their toxicity and so on[29]. So to help improve

the chemical and physical interactions with the body the shortened single walled

carbon nanotubes are derivatized. If un-derivatized, the surface of the shortened

single walled carbon nanotubes is inert chemically and as such it is incompatible

with almost all organic and inorganic solvents, which in turn makes them less

adaptable for future uses and applications [29]. So in short the the overarching

goal for the single walled carbon nanotubes is to increase their solubility in water,

make them more biocompatible, and decrease the toxic effects [29].

The shortened single walled carbon nanotubes can be derivatized by bonding

oligonucleotides, biomolecules, surfactants, and polymers functional groups [29].

It has been reported that after being functionalized, the derivatized single wall

carbon nanotubes solubility increased dramatically; and several studies

have also shown that with increased solubility (or dispersion), the performance of

derivatized single walled carbon nanotubes increases as well as lowers their

toxicity [29]. The derivatized single walled carbon nanotubes also have fantastic

electro-optical properties, high tensile strength, and a high surface-area-to-

volume ratio that also allows for surface derivatization [29].

Other report have also shown that the highly water-soluble derivatized single

walled carbon nanotubes have been absorbed into the cells without any bodily

response [29].  This proves that derivatized shortened single walled carbon

nanotubes are much more biocompatible with physiological systems and as such

much less toxic [29]. With this in mind it is entirely possible to use these

derivatized shortened single walled carbon nanotubes to infiltrate cells without ill

effects, and as such would be perfect to use as trackers or payloads for delivery

to very specific locations [29]. All in all the derivation of the shortened single

walled carbon nanotubes makes them safer and more effective in interacting

with the body and as such easier to be delivered and used as the contrast agent

to the MRI [29].

Shortened carbon nanotubes have several medical applications [11]. They

can be used for imaging of a specific tissue by adding a specific coating on the

surface of the shortened carbon nanotubes consisting of antibodies to target

specific tissues [30]. Single walled carbon nanotubes can also be used to treat

cancer, such as breast cancer [30]. A modified single shortened carbon nanotube

is functionalized for tissue targeting and is filled with an anticancer drug

doxorubicin that is released once the carbon nanotube enters the cancerous cells

[30].

We can improve the invention in [21] on several levels to use for treating

breast cancer [30]. In invention in [30], shortened carbon nanotubes are coated

with polysaccharides, sodium alginate (ALG) and chitosan (CHI) [30]. The

modified short single wall carbon nanotube contains doxorubicin as a cargo,

which is an anticancer drug [30]. The DOX-CHI/ALG-SWCNT includes folic acid

(FA) on its surface as a targeting group for cancerous tissue [30]. The

polysaccharides ALG and CHI are used because of the increased

biocompatibility they confer to the carbon nanotubes [30]. The modified single

wall carbon nanotubes obtained have an improved ability to accumulate in

specific cancerous tissue and to release the toxic doxorubicin in a controlled

manner [30].

To prepare the ALG-SWCNTs, 20 mg of the shortened carbon nanotubes

are sonicated in a sodium ALG solution for 20 minutes [30]. The sodium ALG

solution is made from 40 mg of sodium alginate in 40 ml of 0.1M aqueous NaCl

[30]. After sonication, the solution is stirred for 16 hours at room temperature

[30]. The modified carbon nanotubes obtained are then collected and purified by

ultracentrifugation using ultrapure water [30]. The goal of this purification process

is to remove any unbound ALG [30]. The modified carbon nanotubes are finally

dried at room temperature to obtain the ALG-SWCNTs [30]. Next, 10 mg of ALG-

SWCNTs are sonicated for 20 minutes [30]. Then a 20 ml CHI solution made of

20 mg of chitosan in 0.1M aqueous NaCl and 0.02M acetic acid is added to the

ALG-SWCNTs [30]. The ALG-SWCNTs and CHI solution are stirred at room

temperature for 16 hours [30]. Then after ultracentrifugation, purification and

drying, we obtain CHI/ALG-SWCNTs [30]. Afterwards, 4 mg of CHI/ALG-

SWCNTs are suspended with 6 mg of FA (folic acid) in an 8 ml pH 7.4 PBS

buffer solution [30]. 5mg of EDC.HCl is then added and the mixture is stirred at

room temperature for 16 hours [30]. Following the same steps as described

above we obtain FA-CHI/ALG-SWCNTs [30].

Finally to load the doxorubicin into the carbon nanotubes, 9 mg of DOX

hydrochloride is stirred with 3 mg of FA-CHI/ALG-SWCNTs dispersed in 6 ml of

PBS buffered solution of pH 7.4 at room temperature for 16 hours [30]. Following

the same steps the final functional modified single wall carbon nanotubes is

obtain and can be used to deliver the drug [30].

Shortened Carbon Nanotubes

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