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Vanderbilt University
Department of Biomedical Engineering
A Design for Using Paramagnetic Nanoparticles as
a Cancer Treatment Technique
April 22, 2003
BME 272-3 Group 15
Members : Ashwath Jayagopal
Sanjay M. Athavale
Amit K. Parikh
Project Advisor : Dr. Dennis Hallahan, VUMC
Course Instructor : Paul King, P.E., PhD
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Abstract
Cancer is one of the leading causes of death in the United States, with over 16
million people having been diagnosed since 1990. While a number of treatment methods
currently exist, their disadvantages are numerous. Therefore, we sought to design a
system that encompasses paramagnetic nanoparticles constructed of iron oxide, in
conjunction with radiation processes and multiple degradative enzyme coatings, which
can be delivered to a tumor directly and guided within the tumor matrix.
The
methodology of our design process was systematic, precise, and could be broken down
into two stages. Stage one involved the design of the nanoparticle through extensive
research and advising from numerous resources stated previously. The second stage
encompassed testing the validity of the nanoparticle-enzyme delivery system, with
numerous test runs in a model Matrigel tumor matrix, as well as inspection via Raman
spectroscopy. Our results showed that the uncoated nanoparticles were unable to traverse
through the Matrigel tumor matrix. As for the nanoparticle-enzyme design that utilized
collagenase-H, it was found that after 24 hours, the nanoparticles had successfully moved
moderately through the tumor matrix, which was a primary design objective. In the case
of the nanoparticle-enzyme design that utilized both the collagenase-H and α-amylase
enzymes, degradation of polysaccharides as well as collagen led to larger openings
through which the nanoparticles could disperse successfully.
The Raman spectrum
showed that there are significant differences between the tumor matrix before and after
addition of the nanoparticles, meaning that the integrity of the tumor matrix had been
compromised. In this report we have demonstrated the development of a design which
has been shown to enhance the delivery of magnetic nanoparticles through an in vitro
tumor matrix model. Our findings show that our nanoparticle-enzyme system penetrates
the matrix by degrading tumor structures over time given an external magnetic field. As
we consider the potential of this design, we realize that there are a wide variety of issues
to be addressed. These include effectiveness of our system in vivo, as well as the
nanoparticle type and tendency to clump, effects of nanoparticles in the body for
prolonged periods, and the effects of radiation when applicable.
This design has
tremendous market potential, with direct applications to inhibition of tumor angiogenesis
and tumor development, and could be an alternative to radiation therapy or
chemotherapy.
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Introduction
Cancer is one of the leading causes of death in the United States, with over 16
million people having been diagnosed since 1990. Additionally, over 1.3 million people
are expected to be diagnosed this year, with a 5 year survival rate of under 62% [1].
While a number of treatment methods currently exist, a wide range of disadvantages are
associated with them.
Chemotherapy, radiation therapy, and surgery are common
techniques of treatment. Chemotherapy and radiation therapy methods frequently affect
healthy tissue in addition to cancerous areas, which can cause a variety of side effects.
These can include relatively minor problems such as nausea, vomiting, hair loss, or major
problems such as immune system weakness and low blood counts [2].
Radiation
exposure is the cause of many problems in the body. Surgery is not always an option and
may have side effects depending on the type of operation. Cancer medications in the
circulation can affect the entire body. In addition, adjuvant therapies, or pre-emptive
operations to increase the effectiveness of later procedures, require additional costs and
visits [1]. Severe pain, loss of productivity, and overall discomfort is associated with
cancer treatment today. In light of these disadvantages, much current cancer research is
focused on designing site-specific treatment methods that avoid healthy tissue and attack
a tumor within, destroying it from the inside out. One of the most promising approaches
involves the inhibition of tumor angiogenesis, or the growth of blood vessels [3].
In understanding the research that is directed towards this arena, it is important to
observe the characteristics of a tumor and its internal structure. A tumor’s internal
structure, referred to as a matrix, is a fibrous maze of blood vessels, tumor cells, and
connective tissue. Many proteins contribute to this structure. It is difficult to deliver
medication to a tumor matrix due to these structures, which serve as barriers [3]. As we
wish to target the tumor cells within the matrix, which give rise to the surrounding tumor
vasculature, it is important to visualize the cells’ interaction with the basement
membrane. The interaction of tumor cells with the basement membrane is an important
factor in the regulation of cell behavior. The extracellular matrix (ECM) of the tumor that
we have described is secreted by tumor cells to form an interstitial basement membrane
that forms the framework to which cells are attached. The basement membrane separates
cells from connective tissue, and provides spatial orientation and stability required for the
growth and differentiation of cells. The ECM has also been implicated in the
sequestration, storage and presentation of growth factors to tissues [4]. The basement
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membrane, as well as the endothelial cells attached to it, are viewed as significant barriers
to direct tumor invasion treatment. An effective cancer treatment process that is tumor
site-specific must be able to penetrate the basement membrane to reach the matrix.
We sought to design a system that encompasses paramagnetic nanoparticles
constructed of iron oxide, in conjunction with radiation processes and multiple
degradative enzyme coatings, which can be delivered to a tumor directly and penetrated
within the tumor matrix. We wished to control this system by means of an external
magnetic field. This system was to be designed for cancer treatments that involved tumor
angiogenesis inhibition, tumor blood vessel blockage techniques, drug delivery methods,
or local radiation delivery methods by means of radioisotopes. Our overall goal was to
increase the comfort of the patient, improve the potential for survival by minimizing the
harm of healthy tissues while targeting cancerous areas, increase the available scope of
treatment methodologies, or to at least progress significantly in these aspects. We now
discuss some background on the primary tools used in our methodology.
Paramagnetic nanoparticles made of iron oxide were demonstrated to be capable
of precise control with the application of a magnetic field [5]. In addition, their size can
be up to 100,000 times smaller than the width of the human hair, making biomedical
applications increasingly possible [6]. Current research by Dr. John Zhang at Georgia
Tech is concerned with developing a variety of nanoparticles of various sizes, chemical
compositions, and magnetic properties, for a variety of purposes. They can be massproduced to create thousands of identical nanoparticles whose behavior is predictable.
Essentially, Zhang’s goal is to create a “recipe book” of magnetic nanoparticles for
biomedical, electrical, and aerospace use, among many fields.
These interesting
properties of nanoparticles, supported by our advisor Dr. Dennis Hallahan’s suggestion of
using magnetic nanoparticles in a drug delivery capacity, led to our decision to begin
observing their properties for the purpose of cancer treatment.
While considerations were made to conduct nanoparticle-tumor interaction studies
using in vivo methods, we found that a manufactured tumor reconstruction medium
existed. This in vitro model would allow for us to conduct multiple trials without the
risks and complications associated with mice. BD Clontech’s Matrigel was documented
as being a primary tool in studies on tumor invasion and angiogenesis studies. The
reconstituted basement membrane Matrigel matrix is derived from the Engelbroth-HolmSwarm (EHS) mouse sarcoma, which has been found to be a rich source of ECM
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proteins. The BD Matrigel Matrix is composed of laminin, collagen IV, nidogen/enactin
and proteoglycan - a composition comparable to basement membrane proteins. The
Matrigel matrix has been found to induce differentiation in a variety of cell types such as
hepatocytes, mammary epithelial cells and endothelial cells [3,4]. When endothelial cells
are cultured on Matrigel, they are induced to form capillary tubes within 24 hours [4]. In
essence, the cells resemble in vivo activity, in a tumor that is in vitro. Matrigel was
chosen as a test model for our system as it accurately and rapidly induces angiogenesis
and is a cost-effective, convenient option to animal subjects.
Methods
Currently the field of magnetic nanoparticle drug delivery is in its infancy, yet
holds vast potential. Therefore, the methodology of our design process was systematic,
precise, and covered all areas of interest including safety, design, and testing issues. The
Innovation Workbench software facilitated our thought process, and is shown in the
appendix to indicate the depth of our approach. Our method can be broken down into
two stages. Stage one involved the design of the nanoparticle through extensive research
and advising from numerous resources stated previously. The second stage encompassed
testing the validity of the nanoparticle-enzyme delivery system, by way of numerous test
runs in a model tumor matrix, as well as inspection via Raman spectroscopy.
The particles which we used, created by
FeRx™, are produced by mechanically milling a mixture
of iron and carbon (95% iron, 5% carbon) powders,
without application of external heat. As seen in Figure
1, the nanoparticles have an iron core, with an activated
carbon coating. Previously, the carbon coating served as
Figure 1 – FeRx™ nanoparticles with an
iron core and carbon coating
a means by which to adhere specific antibiotics to target
the tumor vasculature.
However, the same activated
carbon coating also caused a significant amount of clumping, due to C-C stretching bonds
between nanoparticles. Thus, what was once a 500 nm particle becomes a 1-10 micron
particle, increasing the inability of the particles to properly disperse through the tumor
matrix.
Current research in the field of nanoparticle drug delivery focuses on decreasing
the size of the nanoparticle in order to increase mobility through the tumor vasculature.
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However, as the size of the particles decrease, so does their magnetic dipole. Our design
employs a previously uninvestigated area of nanoparticle drug delivery that takes
advantage of the activated carbon coating of the FeRx™ nanoparticles. Rather than
trying to alter the physical structure of the nanoparticles by making them small enough to
permeate the tumor matrix, we decided to adhere them to specific proteases. These
proteases would essentially "cut" the tumor matrix, allowing for free passage of the
nanoparticles.
The design of our project involved a vast amount of research and guidance from
numerous outside sources. It should be duly noted that the steps used in the following
paragraphs are in fact a design, not a mere synthesis of research protocols based on
existing tests. These steps were carefully thought out and calculated, and have not been
documented to have been done to date.
To begin our design, the FeRx™ nanoparticles were washed three times in
Phosphate Buffered Saline solution (PBS) and vortexed between each wash. This was
done to ensure that excess degraded particles and other impurities were removed. PBS is
used for cell washing, as a diluent for media or assays, or as an inorganic base in standard
media. PBS combines inorganic salts, which maintain physiological pH, osmotic
equilibrium, and membrane potential [7]. After washing the nanoparticles in the PBS
solution, they were again washed in 100% ethanol in the same manner three times. The
purpose of the ethanol wash was to decrease the clumping that plagues most nanoparticle
solutions. By dissolving the nanoparticles in a liquid adsorption medium (ethanol) that
includes excipients selected to minimize the hydrophobic Van der Waals forces between
the particles, agglomeration of the particles in the medium is prevented [8].
Next, one mL of the washed particles was removed and placed at –20 ˚C. The
remaining two mL of nanoparticles were then ready for enzyme adhesion. Two specific
enzyme solutions were used in our nanoparticle-enzyme design.
The first enzyme
solution included collagenase-H (Sigma-Aldrich, Collagenase from Clostridium
histolyticum). Collagenase-H was chosen due to the fact that it degrades collagen, a key
structural protein found in the basement membrane of all tumors. Collagen is a form of
connective tissue that is one of the key inhibitors of nanoparticle movement through the
tumor matrix, as mentioned previously. The other enzyme solution included collagenaseH as well as α-amylase (Sigma-Aldrich, Amylase from Cl. hist.). Amylase, like collagen,
is a degradative enzyme that breaks down polysaccharides. Polysaccharides are large
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polar molecules that serve as a structural barrier to the movement of nanoparticles as
well.
Enzyme adhesion was performed in two different sample sets, as depicted in
Table 1. The first sample set (1 mL by volume) was soaked in a 0.1 g/ml solution of
collagenase-H. The second set (1 mL by volume) was soaked in 0.1 g/ml solution of
collagenase-H and α-amylase. Both sets were allowed to soak at room temperature for
Table 1 – types of nanoparticles designed, classified by type of enzyme used.
three hours in order to allow for proper adhesion of the enzymes to the nanoparticles, as
shown in Figure 2. At the end of the three hour period, 0.5 mL of each sample set was
removed and stored at –20 ˚C, for later assessment via Raman spectroscopy. Once all
nanoparticle-enzyme samples were completed, they were then ready for placement in the
Matrigel tumor matrix. Stage one of the design project was complete. The nanoparticles
had been successfully created and were now
ready for application and testing within the
Matrigel tumor matrix.
The first step in stage two, or the testing
phase, was making the Matrigel tumor matrices
better suited to simulate an actual tumor. The
Figure 2 – nanoparticles-enzyme design, which takes
advantage of the activated carbon coating
Matrigel tumor matrix was grown in petri dishes
with a diameter of 8 cm. Within the Matrigel
tumor matrix, endothelial cells were grown in order to more accurately simulate the
physiological makeup of a tumor's basement membrane. In our specific case, we used
generation
six,
human
endothelial
cells
at
a
concentration of 600 cells/mm2.
At this point the Matrigel tumor matrices were
ready to test the validity of the nanoparticle-enzyme
design.
The first step in treating the tumors was
irradiating them with x-rays. The standard irradiation
dosage for tumors 8 cm. in size is 4 Gy [10]. Generally,
the dosage of irradiation for humans runs in the range of
Figure 3 – initial injection of
nanoparticles into the Matrigel
tumor matrix
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50–60 Gy, depending on the severity of the tumor. In our case, the tumor was irradiated
for 2.11 minutes. Radiation was included as part of our design process to complement
nanoparticle delivery, as it was observed in early testing to enhance the permeability of
the tumor vasculature. A small set of Matrigel samples were not irradiated, for the
simple purpose of observing our system’s progress without the aid of radiation. We used
a relatively small dose of radiation as our goal was to avoid the undesirable effects of
radiation. We theorized that a small dose of radiation would be sufficient to slightly
enhance nanoparticle tumor mobility, while not exposing the tissue to excessive
radiation.
Standard radiation oncology lab procedures were complied with, with
supervision from Vanderbilt University Medical Center personnel.
For six matrices, the ordinary, washed nanoparticles were injected, as seen in
Figure 3. The side of the matrix in which the nanoparticles were injected was marked
with a pen.
For six separate matrices, the nanoparticle-enzyme combination with
collagenase-H only was injected and properly marked. For the final six matrices, the
nanoparticle-enzyme combination with collagenase-H as well as α-amylase was injected
and properly marked. All samples were placed in a 37 ˚C incubator for 24 hours. During
that time period, a .5 Tesla static magnet was used to guide the nanoparticles through the
tumor matrix. A simplified flowchart of the design process can be seen in Table 2. In
addition, progress assessment of the nanoparticles’ mobility through Matrigel was
performed with photographs.
The final part of stage two involved assessing the validity of the nanoparticleenzyme design, as well as assessing the nanoparticleinduced alterations to the tumor matrix by way of
Raman Spectroscopy.
Raman spectroscopy is the
measurement of the wavelength and intensity of
inelastically scattered light from molecules. The
Raman scattered light occurs at wavelengths that are
shifted from the incident light by the energies of
molecular vibrations. The mechanism of Raman
scattering is different from that of infrared absorption,
and Raman and IR spectra provide complementary
information. Typical applications are in structure
determination, multicomponent qualitative analysis,
Table 2 – simplified design setup of nanoparticles
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and quantitative analysis [11].
Two samples were used in each study. In the case of the nanoparticles, the first
sample was the 0.5 mL portion of nanoparticles that had been washed three times in PBS
buffered solution and then three times in ethanol, as mentioned previously. This sample
was used as our control. The second sample was the nanoparticle-enzyme design that
used collagenase-H and α-amylase as the degradative enzymes. The Raman setup for
testing the nanoparticles can be seen in Figure 4. A diode laser with a wavelength of 785
nm was used at 80 mW of power. The probe was placed against the sample cuvette
containing the nanoparticles, and an integration time of 10 seconds was used.
A
Nitrogen-cooled CCD camera was used to collect the data. In addition, it was very
important that the room the data was collected in was completely dark in order to avoid
background noise from destroying the signal.
The experimental setup for
assessing the Matrigel tumor matrix
via Raman spectroscopy was very
similar to that seen in Figure 4, the
only
difference
being
in
the
application of the probe to the
Figure 4 – Raman experimental setup for acquisition of data on nanoparticles and tumor matrix
sample. In this case, two samples
were also used. The first sample
was that of unaffected Matrigel
tumor matrix. A small segment was excised from a
petri dish and placed on aluminum foil. The probe,
using the same parameters as that seen in Figure 4,
was placed in contact with the sample, and the Raman
spectrum was taken.
The second sample was a
portion of the Matrigel tumor matrix that had been
subjected to the collagenase-H and α-amylase coated
nanoparticles. A small portion was excised from the
center of the Matrigel tumor matrix and a Raman
signal was obtained. Both sets of signals were later
assessed qualitatively using fluorescence reduction
methods and FFT to remove noise.
Figure 5 - Nanoparticles lacking adsorbed
enzymes fail to move through the tumor matrix
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Results
As seen in Figure 5, the uncoated nanoparticles were unable to traverse through
the Matrigel tumor matrix. This result was similar for all 12 runs in which uncoated
nanoparticles were used. This is consistent with numerous other studies that document
the inability of nanoparticles to disperse through a tumor matrix due to its web-like
structure, a key problem in the development of nanoparticles for drug delivery tasks [13].
The problems associated with clumping were also evident in this case, due to the fact that
the nanoparticles were unable to move.
Figure 6 – Adhering collagenase-H to the nanoparticles increased movement through the Matrigel tumor matrix.
As for the nanoparticle-enzyme design that utilized collagenase-H, Figure 6
shows that there is in fact some movement of the nanoparticles through the tumor matrix.
It was found that after 24 hours, the nanoparticles had successfully moved moderately
through the tumor matrix, which was a primary design objective. It is evident that the
nanoparticle-enzyme solution involving collagenase-H was able to partially degrade and
subsequently traverse the tumor matrix. It was crucial that the nanoparticle-enzyme
combination was allowed to remain within the tumor matrix for 24 hours due to the
relatively slow rate at which collagen degradation takes place. The collagen molecule is
a triple helix, which contains the repetitive sequence -Pro(Hyp)-XGly-Pro(Hyp)-, where
X is a neutral amino acid. Collagenase is specific for the X-Gly bond in this sequence,
and is thus also able to cleave denatured collagen and other polypeptides with a related
primary structure [13].
It is also worth noting that Figure 6 shows clumping was
somewhat reduced due to the washing of the nanoparticles in 100% ethanol solution,
which was another primary design objective.
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As for the nanoparticle-enzyme design that utilized collagenase-H and α-amylase,
it proved to be extremely successful, as we had hoped for in our original design. Figure
7 clearly shows the extent of nanoparticle movement during the 24 hour period.
Degradation of polysaccharides as well as collagen led to larger openings through which
the nanoparticles could disperse, thus allowing for a more homogenous distribution
throughout the tumor matrix, most importantly the tumor core. In addition to high levels
Figure 7 – Adhesion of collagenase-H and α-amylase to the nanoparticles lead to a homogenous distribution
of movement throughout the tumor matrix, less clumping was evident from 10 of the 12
runs, which was a direct result of ethanol washing. This has key significance for drug
delivery applications, since it is desired that a given concentration of drug be evenly
distributed throughout the tumor for maximum effectiveness. In order for a tumor to be
destroyed, no living tumor cells can remain. Any living tumor cells quickly metastasize
to create a new tumor, leaving treatment ineffective and repetitive.
As
Raman
for
the
spectrum,
Figure 8 shows that
there are significant
differences between
the Raman spectrum
of the tumor matrix
before
addition
and
after
of
the
nanoparticles.
This
could only mean that
Figure 8- Raman spectrum of tumor matrix before and after
addition of the nanoparticles.
the integrity of the tumor matrix had been
compromised as the nanoparticles moved
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through the matrix. The Amide I region of collagen, which has a characteristic Raman
shift at 1650 cm-1, had a much higher intensity in the tumor matrix prior to the
application of the nanoparticles.3
In addition, the CH2 bend indicative of collagen
structure, found at 1450 cm-1, was much stronger in the tumor matrix prior to the
application of the nanoparticles. Both of these fingerprints verify that the tumor was in
fact degraded as the nanoparticles moved through the tumor vasculature and endothelial
cells. This adds to the validity of our design, in that the nanoparticles were much more
mobile with their enzyme coating. The final fingerprint which proved that our design
was successful was the Raman shift at 1050 cm-1, which is characteristic of the organized
structure of polysaccharides.
The signal drops significantly between pre and post
nanoparticle addition, which shows the polysaccharides present in the tumor matrix were
broken down. This is very important for successful movement of nanoparticles through
the tumor matrix, being that polysaccharides are large polar molecules that significantly
hinder the movement of nanoparticles through the tumor matrix.
As for the Raman spectrum of the nanoparticles, the FeRx™ particles seemed to
have successfully absorbed the key enzymes necessary for breakdown of the tumor
matrix. The Raman spectrum for the bound nanoparticles shows characteristic peaks at
1050, 1350, and 1550 cm-1. These peaks correspond to the characteristics of amylase,
collagenase, and the activated carbon coating respectively. Degradation of the tumor
matrix must have been due to the nanoparticle-enzymes design, based upon the Raman
spectrum.
It was observed that our radiation dosage had a negligible effect on nanoparticle
mobility through the tumor. We had provided only a small radiation dosage, which may
have been too small to affect permeability. The nanoparticle-enzyme combination moved
effectively through the tumor matrix in samples both exposed to radiation and not
exposed to radiation. This is a promising result as we realize that our combination can
degrade the matrix without the aid of radiation. In vivo testing is certainly essential for
developing further beyond this finding.
Having evaluated our results, we have constructed a general layout of what we
believe to be an effective nanoparticle-enzyme system and complementing process to
deliver medication to a tumor.
Ignoring the medication aspect for a moment,
nanoparticles themselves, characterized by clumping, could even be used by our process
to simply block blood vessels within a tumor, to essentially cut off the supply of nutrients
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and oxygen, starving the tumor. Furthermore, research at Vanderbilt University by Dr.
Ling Geng has involved the investigation of biological growth factors, such as TNF
(tumor necrosis factor) and various forms of EGF (endothelial growth factor). These
relate to tumor blood vessel proliferation and overall tumor development.
Our
mechanism could be used to bind nanoparticles to relevant receptors such that tumor
angiogenesis and/or overall development is reduced.
However, only with in vivo
experimentation with a wide variety of cancer samples (which requires a substantial
budget, resources, and research consortium) can we continue development of this
process. On an initial scale, however, our aforementioned concentrations of enzymes,
and our preparation method, can be applied to various types of nanoparticles suited for
specific tumors, which lays substantial groundwork, especially in the potentially
significant field of inhibiting tumor angiogenesis (by inhibition of growth factors) and
tumor cell survival (by blocking vessels). Following is a suggestion of implementation
for the technique we have developed.
It is important to understand that the objective of our design project was to
develop a method for delivering nanoparticles to a tumor and through the basement
membrane. This in itself was a complex task, and was also done in vitro. Therefore, it is
out of our scope to accurately suggest how a procedure that has not been invented could
be performed. Nevertheless, this suggestion provides an idea of how our technique could
potentially be employed following subsequent development, perhaps on the scale of years
of further research in vivo. First, a patient may be locally anesthetized in an inpatient
procedure.
Using various imaging methodologies, the nanoparticles, having been
prepared with our method, would be injected at the site of the tumor. With a physician
trained in this method, the nanoparticles could then be guided by an external magnetic
field to the tumor site. The timing of tumor matrix degradation is on the scale of hours as
we have shown; thus, the magnet may need to be placed in various areas for a long time
depending on the size and location of the tumor. It is important to note that while our
experiments handled a tumor at the surface directly, skin and adipose tissue serve as
factors that distance the magnet from the nanoparticles and their target, which may
dictate the need for stronger magnetic fields. As magnets are currently used for the
purpose of relieving pain, this fact has been addressed in many cases.
Having suggested a general procedure for implementation, we now address
several safety concerns. The designsafe document in the appendix was generated to
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guide our design process in a manner such that safety was a priority. However, as this
project stands at the beginning of a developing field, it is out of the scope of our design to
address all of the potential risks that could be involved in the process. In fact, it is likely
that many additional hazards than those mentioned here may develop along with the
technique of cancer treatment with nanoparticles. Nevertheless we consider important,
initial considerations here. To begin, we are certain that the magnetic fields involved in
guiding these nanoparticles are not harmful to the human body. Our magnetic field is
orders of magnitude less than MRI, which is also known to be a safe biomedical
application of magnetic fields. We did not incorporate an electromagnet in our design,
eliminating any risk associated with electrical hazards. Aggregation of nanoparticles
within the human body can have harmful consequences, such as obstructing blood flow.
It is important, therefore, that the nanoparticles are to a certain degree paramagnetic
(slightly magnetic) and consist of a low energy barrier, such that they are less likely to
clump with a highly-varying magnetic field.
Our nanoparticles exhibited this
characteristic somewhat, though not to the extent which we had originally hoped for. A
variety of nanoparticles may have been available that would have been less likely to
clump.
It is for this fact that several companies, such as Chemicell, Gmbh, and
Nanomag, as well as researchers at Georgia Tech led by Dr. John Zhang, are primarily
devoted to developing nanoparticle “recipes” consisting of various properties for several
biomedical applications. They emphasize the fact that coatings on nanoparticles, as well
as standard protocols for manufacturing need to be developed that address this problem
directly, before deeply pursuing in vivo studies. Our specific solution to this problem was
the use of ethanol in our method of preparation, which slightly reduced clumping
compared to nanoparticles not coated with ethanol. Another concern is that once in the
body, the nanoparticles must avoid immune system reactions, yet remain in the
circulation for a long enough time if it is necessary. Disguising the nanoparticle as an
albumin protein, or coating it with certain biological factors is a technique which must be
addressed in future research.
Our thought on this problem was that since the
nanoparticles are injected into the tumor directly (or near it), this may not be a problem in
some cases. However, if the nanoparticles do not degrade within the body, then problems
could occur. This should be the focus of another research activity. Our nanoparticleenzyme system was designed with biocompatibility as a priority, as only the iron oxide is
a substance that is not found in great amounts in the human circulation. In addition,
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whenever radiation methodologies are involved there are serious risks presented. From
our prospective, should our system be used to deliver local radiation, as was the case with
Iversen and Falk at Vanderbilt University in 2002, it would be important to use low halflife substances to reduce the risk of exposure. As we are not convinced yet that radiation
was significant in our system’s effectiveness, we cannot be sure of whether radiation will
always be needed for the purpose of enhancing nanoparticle delivery to a tumor. On the
other hand, radiation treatment may be needed to treat the tumor regardless, and thus
considering radiation effects would be essential in investigating nanoparticle therapy.
Thus it may be more beneficial to consider our system as a method of inhibiting tumor
angiogenesis or starving the tumor by blocking vessels within it, which may complement
radiation therapy or chemotherapy, or may replace it entirely, depending on how this
technology develops in years to come. Initially we find that our system’s benefits will far
outweigh risks should these problems be addressed effectively.
In light of our qualitative (photographs) and quantitative (Raman) analysis
attesting to the promise of our design, placed side by side with the applications to cancer
treatment (inhibition of angiogenesis, complement to current treatments) we see that the
nanoparticle-enzyme system may have significant implications on the drug delivery
market. The drug delivery market has an estimated worth of $24 billion dollars as of
2002, according to the annual Scrips Report. It is reasonable to state that a number of
pharmaceutical companies may embrace a developed version of our technique, as well as
start-up companies and nanoparticle manufacturing industries, which would deal solely
with this technology, potentially necessitating research expenditures on the order of
millions annually. As this technique could apply to the drug delivery market, it also
applies to the cancer research arena, which received over $4 billion in federally-funded
research alone in 2002, according to the American Cancer Society. We have documented
the statistics on people affected with cancer in the United States. Should this technology
develop and become a successful treatment methodology, our nanoparticle-enzyme
system could potentially affect 1.3 million patients a year diagnosed with cancer in the
US alone, independent of cancer patients worldwide. This in itself is a significant market
prospect. The cost of marketing this technology is difficult to estimate, but would entail
informative and training sessions for physicians, hospital administrators, and nurses.
Our technique is cost-efficient, especially when compared to other treatments
currently used that it could potentially replace. Radiation therapy and chemotherapy
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methods can range from the thousands to hundred thousands of dollars depending on the
specific case, while radiation seed treatment can cost around $2000, and surgical
procedures are usually on the order of $10,000-50,000 according to the American Cancer
Society. While a specific drug and/or chemical treatment, or type of nanoparticle used
may affect our costs of developing our nanoparticle-enzyme system, the additional
budget needed for implementation of our design is still trivial compared to the current
methods. In the future, a physician might order a specific nanoparticle-enzyme solution
and inject it, using existing imaging technologies for guidance in association with
radiation and a magnetic field.
Although it may be premature, we could see this
procedure being less than $10,000, with the specific cancer type and medications varying
that cost profile. In addition, in light of our figures on cancer costs presented earlier, we
would increase work productivity for people living with cancer. A procedure could be as
simple as an outpatient treatment session; since healthy tissue would not be harmed, it is
likely the patient would experience few or none of the side effects associated with
chemotherapy or radiation, which weakens physiological systems and prevents the
patient from participating in any activity whatsoever. It is difficult to perform a direct
benefit calculation as we are not certain if this technology will be implemented in a
biomedical technique, and if it will be used in all cancer treatments or a fraction of them.
However, we can emphasize that the development cost of our system was approximately
$1000.00 in value for materials. This includes enzymes and Matrigel dishes, as well as
FeRx nanoparticles and Matrigel solution. For the preparation of what we believe to be a
standard tumor dosage of an EHS tumor (that which is reproduced in Matrigel), it would
cost 3 hrs * $15/hr = $45 labor. As the person preparing this solution would be a trained
lab technician, these costs are close to accurate. The maintenance costs of our system
could be high as they involve a temperature stabile environment for the solution to
maintain its integrity.
An important aspect of this technique’s development would
certainly involve FDA proceedings, which would initially investigate our process in
terms of necropsy tests and toxicology tests to assess initial risks. As we have yet to
implement the system in vivo, there are multiple considerations that we believe the FDA
would consider, and several phases of animal testing would need to be conducted for
potentially several years before this technology is introduced.
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Conclusions
In this report we have demonstrated the development of a design which has been
shown to enhance the delivery of magnetic nanoparticles through an in vitro tumor matrix
model. Our findings show that our nanoparticle-enzyme system penetrates the matrix by
degrading tumor structures over time given an external magnetic field. We had initially
sought to develop a system that allows for site-specific nanoparticle delivery with
minimum aggregation to a tumor and its inner structure, with a nearly homogenous
distribution of nanoparticles once inside the tumor. Based on our results we conclude
that we have satisfied desired specifications, although with future work and collaboration
our system could be much more effective. Some problems with this technique that we
have considered are too advanced to address at this time, and should certainly be resolved
in future research efforts, such as immune response, and extended nanoparticle presence
in the body. We consider our design to be an extremely functional process which is
worthy of further development in advanced laboratories and industries worldwide. It is
certainly reasonable to state that this technology will not vanish and will probably be in
widespread use in the next 20 years, for some application that may not be cancer
treatment. For instance, nanoparticles serve as excellent contrast agents in MRI. The
market for this technology is extensive, as the funding ranges in billions and patients
benefiting from this treatment would be millions. Our technique has a potential direct
application in the inhibition of tumor angiogenesis and blocking tumor oxygen and
nutrient intake, and may also be employed in delivering local medication (anti-tumor
drugs) or radiation. We wish to build on our findings throughout this summer and
potentially beyond it, which may result in a publishable work so we can share our
knowledge with others to advance the field of cancer treatment.
Recommendations
As we consider the potential of this design, we realize that there are a wide variety
of issues to be addressed, many of which we have discussed. These include primarily the
effectiveness of our system in vivo, as well as the nanoparticle type and tendency to
clump, effects of nanoparticles in the body for prolonged periods, and the effects of
radiation when applicable. In addition to these, we acknowledge that there are many
enzymes which we did not consider that may enhance the effectiveness of our system. In
fact, a peptide library (which Dr. Hallahan has been investigating at Vanderbilt for use
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with liposomes) that binds to corresponding sites might be used with nanoparticles to
enter a tumor via a lock-and-key mechanism. Cell-mediated transport is an important
arena which is certainly worth investigating in conjunction with this technology. We
would also recommend that radiation and its effects on our method are worthy of study.
Radiation remains a predominant cancer treatment method, and will likely continue to be
one, and thus current technologies must be considered in light of developing techniques
in order to consider the most effective process. MRI control of magnetic nanoparticles is
not impossible, though the magnetic fields generated by MRI cannot be harnessed to
precisely control nanoparticles as that is not its function. It is very important that the
procedures followed in the design and implementation of our system be repeated many
times to ensure that our results were accurate, and also to perhaps make new discoveries,
and enhance our understanding of the system. A variety of specialists, in radiation
oncology, radiology, physics, electromagnetics, and chemistry, to name a few fields,
should read this report and provide feedback. As we have received very useful feedback
up to this point from our advisors and other Vanderbilt University personnel, we are
certain that feedback is essential in this design’s success in the future.
As participants in this design project, we feel it is our ethical responsibility to a.)
share our findings and design with those who can help us improve it, and b.) to continue
advancing this design, and not abandon it.
Many people would benefit from this
technology if implemented in the future, and we are excited to have taken part in it and
look forward to continue developing this technique. We have demonstrated throughout
this report that our process is worthy of further development in research institutions and
industries, and with collaboration of personnel and resources each of the issues facing
advancement of this technology can possibly be handled with success.
Works Cited
[1] American Cancer Society “Cancer Facts and Figures 2003,” www.cancer.org
[2] National Cancer Institute “A Guide to Self-Help during Cancer Treatment.” 2001.
[3] BD Clontech. “Matrigel Professional Guide to Protocols.” 2002 P. Dasgupta,
Columbia Univ. www.bd.com
[4] Matrigel Product Specification Sheet, www.bd.com, 2003
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[5] Lee, Westerval, et al. Microelectromagnets for the Control of Nanoparticles. J Appl
Phys, vol 79, 2001
[6] Shuming Nie, Georgia Institute of Technology, with Dr. John Zhang. Commentary
on Nanotechnology.
[7] http://www.cellgro.com/catalog2/results2.cfm?ID=16
[8] http://www.pharmcast.com/Patents/2002/November/64_Magnetically111902.htm
[9] Challa Kumar, Carola Leuschner, Josef Hormes and William Hansel, "Magnetic
nanoparticle bound lytic peptides and their conjugates as potent anticancerous drugs
as well as drug delivery systems", A patent being filed.
[10] O’Reilly M. S., Holmgren L., Shing Y., Chen C., Rosenthal R. A., Moses M., Lane
W. S., Cao Y., Sage E. H., Folkman J. Angiostatin: a novel angiogenesis inhibitor
that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 79:
315-328, 1994
[11] Introductory Raman Spectroscopy by John R. Ferraro and Kazuo Nakamoto
(Academic Press, 1994).
[12] http://www.serva.de/products/knowledge/061332.shtml
[13] E-H-S (Engelbreth Holm Swarm tumor (mice)) Matrigel Experimental
Considerations, 2003; www.bd.com/
Please email [email protected] for any information.
Appendix
1.) Designsafe Analysis
2.) Innovation Workbench Process