Improving Cancer Therapy: Triggering Chemotherapy Using Temperature
Responsive Polymers
Mary Kathryn Sewell
Christopher S. Brazel, Ph.D.
Professor of Chemical and Biological Engineering, The University of Alabama
Abstract:
A combination cancer treatment platform
that combines hyperthermia and chemotherapy was
investigated. By including magnetic nanoparticles
in a thermally responsive hydrogel, a smart drug
delivery system was created. Drug delivery was
triggered using in vivo experiments to simulate
hyperthermia treatment.
The combination of
treatment methods could improve cancer therapy
efficacy and decrease negative side effects currently
associated with cancer treatments.
To create an even heating profile,
nanomagnets must be uniformly dispersed within a
hydrogel. The size of agglomerations of FePt
nanoparticles was dependent on the initial
concentration of nanomagnets. Drug diffusion
coefficients were determined for a model drug,
theophylline, when released from thermallysensitive
poly(N-isopropyl
acrylamide-coacrylamide) gels. From these experiments, the
effects of temperature and FePt concentration on
diffusion coefficients were determined. Increased
nanomagnet concentrations did not obstruct
theophylline diffusion, therefore drug carriers can
be designed without concern about altering their
microstructure due to the nanoparticles. Increasing
temperature above the normal human body of 37oC
to simulate hyperthermia, however, caused slower
release rates in gels that had been dried before the
release studies.
Introduction and Background:
The most common forms of cancer
treatment include chemotherapy, radiation, and
surgery, and to a lesser extent, hyperthermia.
Hyperthermia is a heating therapy in which the
cancerous tissue is heated between 43oC and 45oC
(Kim et al.). The higher temperatures kill tumor
cells because the cancerous tissue cannot cool itself
as quickly or efficiently as healthy tissue.
Hyperthermia is usually used in combination with
other therapies to increase treatment efficiency
(NCI). Localized hyperthermia is limited to the
affected area of a patient’s body; the heating source
may be external or internal (NCI). Chemotherapy
involves treating patients with anti-cancer drugs;
these drugs can either destroy the cancerous cells or
slow their growth (NCI). The high concentrations
of anti-cancer drugs used in chemotherapy can harm
healthy cells as well, causing hair loss, nausea, and
other negative side effects (NCI).
Radiation
treatment uses ionizing radiation to shrink tumors
and destroy cancer cells (NCI). This treatment is
often used in combination with surgery or
chemotherapy (NCI). Surgery is another common
treatment method in which the tumor is removed
from the patient; however not all tumors can be
safely removed.
A metastasis occurs when
cancerous cells spread from the primary affected
region to another region or system in the body
(NCI). Often these metastatic cells cannot be
detected until other tumors have formed (NCI).
A unique solution to the challenge of
effective cancer therapy with minimal side effects is
proposed. Our approach to combine hyperthermia
with chemotherapy utilizes magnetic nanoparticles
in thermally responsive hydrogels.
The
nanoparticles are designed so that an externally
administered AC magnetic field will heat them,
causing the thermal response of the gel. The mesh
network of the hydrogel will change, releasing a
controlled amount of anti-cancer agent at a specific
location. The inclusion of adenoviruses or other
biological agents would allow for targeting of the
treatment. The combination of hyperthermia and
chemotherapy should increase treatment efficacy
(Ito et al.). The targeting and triggering of the
therapy should localize both the heating and
chemotherapeutic agent to help decrease negative
side effects associated with current cancer treatment
options.
This research project is focused on synthesis
and characterization of magnetic nanoparticlecontaining hydrogels to determine their thermal and
diffusive behavior. One objective is to create an
even dispersion of magnetic nanoparticles into the
hydrogel. This will allow for even heating profiles
during hyperthermia treatment. Another objective
is to determine the effect of temperature and
nanoparticle concentration on diffusion of a model
drug from hydrogel systems. A final objective is to
investigate the parameters of magnetic fields that
can successfully heat hydrogels containing
magnetic nanoparticles.
Environmentally responsive polymers have
been studied for some time as a way to increase the
efficacy of pharmaceutical administration, improve
drug delivery platforms, reduce administration
frequency, and decrease side effects (Ankareddi and
Brazel, Brazel and Peppas 1999, Schmaljohann).
The unique characteristics of these materials allow
for engineering of desired responses. This
technology has been in use for some time in the
form of enteric coatings on many drug capsules.
These coatings only dissolve in the basic pH of the
intestines, rather than in the acidic pH of the
stomach. Polymers may be responsive to a change
in temperature, pH, concentration, or a combination
of these factors (Schmaljohann). When stimulated
by a change in one of these conditions, an
environmentally responsive polymer will undergo a
phase transition. This changes the conformation of
the system, causing the desired release behavior.
In order to exploit the thermal behavior of
temperature-responsive hydrogel systems, the lower
critical solution temperature (LCST) needs to be
known. The LCST is the temperature at which a
polymer will undergo phase transition and exhibit a
change in volume or conformation. An example of
this behavior can be seen in poly(N-isopropyl
acrylamide), P(NIPAAm).
Below 32oC, a
PNIPAAm gels are hydrophilic and swell with
water. When the sample is heated above this
temperature, it becomes hydrophobic, effectively
“squeezing” the water out of the sample. The
inclusion of hydrophilic
or hydrophobic
comonomers changes the chemical structure of the
gel and alters the LCST (Ankareddi and Brazel, Feil
et al.). This paper explores the use of PNIPAAmbased hydrogels to characterize drug diffusion and
heating.
Because our system will utilize
hyperthermia to trigger drug release, a copolymer
that has an LCST in the range of 42-45 oC is
desirable. An LCST of approximately 45oC can be
achieved by using16.6 mol% acrylamide (AAm) to
increase the hydrophilicity of PNIPAAm
(Ankareddi and Brazel). These copolymers are
termed P(NIPAAm-co-AAm). Another parameter
that can be altered to control drug diffusion is the
polymer mesh size. This can be controlled to some
degree by changing the amount of cross-linking
agent or comonomer used during synthesis.
Materials and Methods:
Hydrogel synthesis was carried out via freeradical solution polymerization.
Quantities of
reactants and solvents were based on the amount of
NIPAAm used in synthesis; all other values were
calculated based on this number. NIPAAm and
AAm were dissolved in a 50:50 weight percent
methanol and water solution, which was used as the
solvent. Methylene bisacrylamide (MBAAm) at
1mol% was used as a cross-linking agent.
Ammonium persulfate at 1wt% was used as the
free-radical initiator. Tetramethylethylenediamine
(TEMED) was used at 1wt% as the reaction
accelerator. For the heating experiments, poly(2hydroxyethyl methacrylate) or PHEMA was used
instead of PNIPAAm, but it was synthesized
similarly to the P(NIPAAm-co-AAm) samples.
Instead of TEMED, sodium metabisulfite was used
as an additional initiator at 1wt% total monomer
weight.
Samples were synthesized by first adding
measured amounts of NIPAAm, AAm, and
MBAAm to a beaker. Water and methanol were
added, and the solution was mixed on a magnetic
stir plate. For 15 minutes, nitrogen gas was purged
through a balloon into the solution. This removed
any oxygen that could act as a free radical
scavenger during the polymerization. After the gas
purge, the initiator and accelerator were added.
After stirring for 1-2 minutes, the solution was then
pipetted between two siliconized glass plates that
were separated by a Teflon gasket and secured with
clamps. The thickness of the gasket controlled the
thickness of the resulting polymer sample.
Polymerization of a P(NIPAAm-co-AAm) sample
usually took 24 hours. For some samples, a model
drug, theophylline, was added at 1 wt% of the
monomer amount, to the mixture with the
monomers prior to polymerization.
If magnetic nanoparticles were included in a
sample, the synthesis procedure varied. In this
study, FePt
magnetic nanoparticles, also
synthesized at UA, were used. The average size of
the particles was 6nm, dispersed in water with
mercaptoundeconoic acid. After the NIPAAm,
AAm, and MBAAm were well mixed with the
solvents, the beaker was removed from the stirring
plate. The magnetic stir bar was also removed from
the solution. The desired amount of FePt solution
was added drop wise to the solution in the beaker.
The resulting solution was stirred under a nitrogen
gas purge. The accelerator and initiator were added,
and the solution was stirred with a glass rod for 1-2
minutes and then plated as described before.
The drug release studies were completed
using
a
Shimadzu
Model
UV-2401
spectrophotometer attached to Distek Model 2100C
USP type II dissolution cells. A sample was taken
from a cell to the spectrometer via a low-flow
peristaltic pump and Tygon tubing. The Kinetics
setting on UV-Probe software was used to collect
drug release data every 10 seconds over the course
of the experiments. The release medium was 1L of
deionized water kept at 37 or 50oC. Absorbance
data were collected at 275nm for 10 hours. Beer’s
Law, which shows a linear relationship between
absorbance and concentration, was used to
determine the amount of theophylline released over
time. The experiments were run in triplicate to
determine reproducibility.
The heating experiments were carried out to
determine the rate of termperature rise and steady
state temperatures reached using an AC magnetic
field. The instrument used for these experiments
was a custom designed hyperthermia chamber built
by Induction Atmospheres. The six turn solenoid
attachment was used with the chamber. The sample
was placed in a glass vial, and the vial was inserted
into the coils for the experiment. The magnetic
field produced in this experiment was 560 Oe, with
a resonance frequency of 231 kHz. A FLIR
Systems ThermaCAM SC2000 NTSC, with infrared
imaging, was used to collect the temperature data at
the center, midpoint, and edge of the sample. Each
experiment lasted 15 minutes, with temperatures
taken every minute. To establish reproducibility,
four trials were run for each sample. A 12mm
diameter PHEMA hydrogel loaded with 0.75 wt%
FePt was used for the experiments.
Results and Discussion:
At all levels of FePt concentration,
agglomerations were seen in the hydrogels. For
lower initial nanoparticle concentrations, the size of
the agglomerates was smaller than in more
concentrated samples (Figure 1).
At greater
concentrations, the color of the hydrogel is
noticeably darker and the agglomerations appear
evenly dispersed. An optical microscope was used
to verify the size of agglomerations in different
samples.
a
b
Figure 1. Microscope image of FePt
agglomerations in PHEMA hydrogels with (a)
0.10 wt% and (b) 0.75 wt% FePt. The white line
scales to 500 m. Although there are more
aggregates in the 0.10wt% FePt sample, they are
small in size than those found in the 0.75wt%
FePt sample.
One sample of P(NIPAAm-co-AAm) at 1.25
wt% FePt was completely non-uniform with
millimeter sized aggregates. This suggests there is
an upper limit to the inclusion and dispersion of
magnetic nanoparticles in hydrogels. Attaining a
completely even dispersion of nanomagnets may
not be possible. Because the nanoparticle solutions
are synthesized in organic solvents, even after
ligand exchanges on the nanoparticles, the aqueous
nature of the hydrogels may cause aggregates to
form at all concentrations.
The drug release experiments were used to
determine diffusion coefficients for theophylline
from the hydrogel systems (Figure 2). These
coefficients were determined using the early time
approximation of the Fickian equation for diffusion
(Brazel and Peppas 2000):
Mt
 Dt 
 4 2 
M
  
1/ 2
M
,0   t
 M

  0.6

where Mt is the mass of solute released at time t,
M∞ is the total mass of solute released, D is the
diffusion coefficient in cm2/s, t is the time in s, and
 is the half-thickness of the dry hydrogel sample in
cm.
At higher temperatures, the diffusion of
theophylline from P(NIPAAm-co-AAm) gels is
slowed (Table 1). This was expected because the
hydrogel has undergone a phase transition and
collapsed in on itself. The inclusion of FePt had
little effect on the release of theophylline. At higher
concentrations the release rate was marginally
slower than at lower concentrations, although the
difference is not significant. Future experiments
will include magnetically-triggered release studies,
using the hyperthermia chamber. Other future
studies will examine the release behavior starting
from pre-swollen hydrogels kept at 37oC, where a
squeezing mechanism is expected to offer higher
release rates at higher temperatures.
Table 1. Diffusion Coefficients for Theophylline
Release from P(NIPAAm-co-AAm) Hydrogels*
37oC
50oC
0 wt % FePt
4.79 ± 0.09
3.28 ± 0.28
0.002 wt % FePt
4.47 ± 0.20
3.37 ± 0.21
0.05 wt % FePt
4.35 ± 0.08
4.09 ± 0.36
0.125 wt % FePt
4.06 ± 0.21
8
3.10 ± 0.24
2
*All values in10 cm /s
Figure 2. The effects of temperature and FePt
nanomagnet concentration on theophylline release
from P(NIPAAm-co-AAm) hydrogels.
In the heating experiments, the magnetic
nanoparticle agglomerations were shown to have a
negligible effect on the overall heating profile
(Figure 3). The gels heated to approximately 31oC
during each experiment. Despite having an uneven
dispersion of FePt in the sample, the heating
appears uniform. This suggests that as long as the
aggregates are fairly uniform in size, the gel will
heat evenly. Future studies will include different
polymers,
an
increased
concentration
of
nanomagnets, and different operating parameters of
the hyperthermia unit with the goal of heating the
sample to temperature effectively used for
hyperthermia (42-45oC).
Figure 3. The radial heating profile of PHEMA
with 0.75 wt% FePt. The dip in the lines was
caused by the camera’s auto-adjust function.
References:
1.
2.
Figure 4. An infrared thermal image of a
PHEMA gel with 0.75wt% FePt during the
heating experiments. The outer yellow rings
correspond to the coils of the hyperthermia unit
surrounding the sample vial (seen from the top).
3.
4.
Conclusions:
The size of FePt agglomerates in
P(NIPAAm-co-AAm) hydrogels can be limited
with careful synthesis procedures. Preliminary
results indicate that even with aggregation of
magnetic nanoparticles, the heating profiles during
hyperthermia experiments are uniform. Further
work is needed to determine the maximum amount
of FePt that can be included in a gel without
creating an uneven heating profile. The drug
release studies indicate that the increased
nanomagnet concentration does not interfere with
drug delivery and that temperature offers a way to
control the rate of release. Future studies will
determine the release rates for gels with different
mesh sizes and higher concentrations of FePt.
Another set of future studies will be completed to
show magnetically-triggered drug release.
Acknowledgements:
I would like to thank the following people
who have supported my project: Dr. Dong Hyun
Kim, Dr. David E. Nikles, Hitesh Bagaria, Lauren
Blue, Kyle Fugit, Andrei Ponta, and Yhni Thai. I
would also like to acknowledge those who funded
my research: the McWane Undergraduate
Fellowship, the John McKinley Creative Project
Award, and the Alton Scott Memorial Fund.
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Mary Kathryn Sewell, from Helena, Alabama, is a
senior majoring in Chemical and Biological
Engineering. For her work with the Magnetic
Biomaterials Research Group, Mary Kathryn has
received the John McKinley Creative Project
Award, a Randall Outstanding Undergraduate
Research Award, and two McWane Undergraduate
Fellowhips. She is also the Assistant Editor for this
issue of JOSHUA.