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Peptide Drug Delivery System for Anticancer Drugs
Abstract
Improving the selectivity and reducing the toxicity of chemotherapeutic agents has proven to be
a difficult simultaneous task to achieve when using a degradable and synthetic polymer backbone for
drug delivery. By utilizing a peptide based drug delivery system, it may be possible to significantly reduce
toxicity to patients while increasing selectivity and drug effectiveness. This study focuses on the
characterization of that peptide. The peptide can be divided into three main parts: ligands used for
targeting, a hydrophobic section on the inside of the three dimensional protein, and a hydrophilic
section that lies on the outside. The hydrophilic section must resist protein adsorption that would
reduce the effectiveness of the protein. A Surface Plasmon Resonance (SPR) detector was used to
determine the amount of protein that adsorbed to four different peptide chains: CV6, CV6N3, CV4G2, and
CV4S2. The SPR directly correlates a change in wavelength of light reflecting off the peptide surface
during protein adsorption to the amount of protein adsorbed on a surface. We found that CV4S2 to be
the most effect of the four peptides at resisting protein adsorption. Future work will include testing
additional peptides for protein adsorption and incorporating these successful peptides into the larger
carrier molecule.
Introduction
“I will respect the hard-won scientific gains of those physicians in whose steps I walk, and gladly
share such knowledge as is mine with those who are to follow.” Learning and adaptation are traits that
are not uniquely human. However, the passage of knowledge and experience from student to teacher
through generations is exclusively a power belonging to Homo sapiens. The previous quote, a line from
the Hippocratic Oath, shows one such area where this passage of wisdom over millennia has given
insights into medicine and the human body that never could have been conjured three thousand years
ago.
Humans are also unique in the fact that they are not only able to care the sick or injured but
they are able to take active steps in the healing of them. More than three millennia ago in ancient Egypt
and China, humans began to practice crude healthcare. Though based on spiritual beliefs as much as fact
or evidence, the notion that being ill was temporary and could be cured took hold. Five hundred years
later, modern medicine as we know it today took root in Greece with the works of Hippocrates. A
rationale, evidence based approach to healthcare evolved and is quite similar to many of the
experiments performed today.
Advancements in the basic sciences such as biology, chemistry, and physics contributed in a
large way to the understanding of the human body. For example, microorganisms were postulated as
early as the sixth century BCE. However, they were not actually discovered until the seventeenth
century CE. For this reason, sciences such as anatomy and physiology advance almost as quickly as the
other sciences will allow it.
Today, our understanding of the human body and the way it works is vast but by no means
complete. Along with that knowledge, the ability to treat fatal, common diseases came quickly.
Conditions such as dysentery, smallpox, and typhoid fever which were often fatal could now be treated
and cured with nearly zero percent mortality. Along with the introduction of hygiene and antibiotics, the
average life expectancy has doubled, leading to a whole new crop of diseases and conditions that were
considered rare just a few centuries ago.
Among these relatively uncommon diseases, cancer has proved to be one of the most fatal. In
2007, nearly eight million people died of complications due to cancer, more than ten percent of all
worldwide fatalities [1]. Significant research over the past several decades has been dedicated to
preventing, treating, and eradicating this disease. Common treatments for cancer include surgery,
radiation, and chemotherapy. Essentially, chemotherapy is the use of any chemical compound to treat a
disease. The word has evolved with one specific connotation in particular: the use of chemicals to treat
cancer. Its development is relatively recent in the scheme of modern medicine, approximately six
decades. Generally speaking, there are two major research topics. Finding medicines to effectively kill
cancerous cells and specifically targeting cancer cells or their support system and not targeting healthy,
living tissue. Current regimens are relatively good at killing cancerous cells. However, they are also
relatively good at killing healthy cells as well. The development of specific targeting drugs is imperative
to increase efficiency the medications and advance the field of chemotherapy.
Background
The human body is overwhelming complex and many times unpredictable. Each element of the
chemotherapeutic drug must be probed for every interaction that takes place within the human body. In
addition, the carrier molecule is just as important as the drug itself and must possess certain qualities.
Among these qualities is the ability to minimally interact with anything other than the predetermined
target. The body, however, is incredibly effective at eliminating foreign materials through a series of
events called the foreign body reaction. Essentially, anything in the blood stream that doesn’t belong is
quickly covered in proteins that spontaneously ‘stick’ to the target. These act as markers for
macrophages which in turn encapsulate and destroy the foreign object. This is highly undesirable for a
highly selective chemotherapeutic drug. Other undesired consequences are also possible. The
spontaneous adsorption of these proteins can also cause inflammation, infection, or thrombosis. The
first step of both of these consequences can be avoided though. By ensuring that the outside of the
carrier molecule is ‘non-fouling’, proteins do not stick to the materials and never trigger the response.
To understand what makes a material non-fouling requires some background material and
understanding.
Any material that is placed inside the body can loosely be called a biomaterial. A biomaterial is
“a nonviable material used in a medical device, intended to interact with biological systems”.
Furthermore, “biocompatibility is the ability of a material to perform with an appropriate host response
in a specific application” [2]. A number of different biomaterials exist, but they can all be divided into
three basic categories: synthetic polymers, inorganic materials (metals, ceramics, carbon) and natural
materials. Drug delivery has traditionally been accomplished through polymeric biomaterials that
encapsulate small molecule chemotherapy drugs which can then be delivered to cancer cells. However,
there are a number of severe disadvantages that polymeric biomaterials possess. Polymers are
compounds that are a string of repeating units called monomers. In many cases synthetic polymers
cannot be degraded or the products of degradation are toxic. Polymers may also contain remnants of
their production process in the form of leachable compounds such as solvents, catalysts, or terminators
that are also toxic in-vivo. Proteins maintain most of the positive aspects of polymers: complex shapes
and structures, wide range of physical properties and simple surface modification. They also eliminate
the toxicity worry of the degradation products. Proteins are a biological polymer where the repeating
subunit is an amino acid. All the proteins in the human body are composed of amino acids which are
everywhere in the body. Thus, there is no toxicity concern when the proteins breakdown over time. The
amino acids are simply adsorbed. Proteins are expressed inside of cells and can be very highly purified.
Therefore, proteins are a group of promising biomaterial for drug delivery.
Proteins are a sub-class of polymers whose basic monomer units are one of twenty one amino
acids. Amino acids are a very diverse group of compounds that all contain at least one carboxylic group,
amine group, and differ in side chains. The different side chains give the amino acids their unique
qualities. They can be positively charged, negatively charged, polar, or non-polar. They may also contain
additional amines, thiols, or hydroxides. Chains of amino acids are referred to as peptides or
polypeptides. Longer chain polypeptides eventually tend to form secondary structure in the way of
helixes or β-pleated sheets. Additional associations may also occur that create tertiary or quaternary
structure. In total, most proteins contain hundreds or even thousands of individual amino acids. The
overall behavior of the protein in physiological conditions is strongly influenced by the bulk properties of
the protein which is in turn influenced by the addition of each individual amino acid. Furthermore, there
is also a unique structure for each protein in the body. Often, the three dimensional structure is
spherical in nature where the “hydrophobic residues preferentially locate ‘inside’ the protein where
they are shielded from water, while the ionized and polar residues are usually on the outside of the
protein and in contact with the aqueous phase” [2]. This arrangement provides a unique opportunity for
protein interactions. The chemistry and surface interactions of proteins in largely dictated by the polar
and charged surface and the bulk properties such as overall charge, density, and molecular weight, are a
function of all the amino acids.
Having established this, it is important to understand what qualities a biomaterial must possess
in order to avoid protein adsorption. Such materials are often called ‘non-fouling’ or ‘low-fouling
materials’. Recent research suggests that the most effective low-fouling materials posses several of the
same qualities: hydrophilic and overall neutral charge [3]. Thus, any designed carrier biomaterial should
posses these qualities. In fact, it is quite easy to design proteins that possess all of these qualities as
evidenced above. Again, this makes proteins promising candidates for drug delivery.
However, once a non-fouling biomaterial has been proposed, the need to validate the
hypothesis arises. How is it possible to prove that designed proteins can be non-fouling? In the second
half of the twenty century a number of methods were created or drastically improved for the purpose of
studying protein adsorption and kinetics. Some examples include: ellipsometry, Enzyme-Linked
Immnosorbent Assay (ELISA), Circular Dichroism (CD), and Surface Plasmon Resonance (SPR).
Essentially, “SPR sensors are thin-film refractometers that measure changes in the refractive index
occurring at the surface of a metal film” and an attached substrate [5]. There are several critical
components that are common to all SPRs: a light source, crystal to refract light, a collection source, and
a glass chip. The glass chip is coated with two thin layers of metal, titanium and gold, where the surface
to be tested is attached. One of the most common methods of detection is change of wavelength
method. Light enters the crystal and passes through the metal surface and monolayer and is refracted to
the detector. The wavelength of the light shifts as protein is adsorbed onto the surface. The total
amount of protein adsorbed is a function of the total shift (the difference) in SPR wavelength before and
after a protein solution is passed over the chip. A pump is used to pass a protein solution over the chip
and simulate liquid flow such as blood. Accurate measurements are highly dependent of the
immobilization of a molecule on the surface and form a self-assembled monolayer (SAM). Figures 1 and
2 give several examples of what a SAMs may look like. This monolayer is representative of the surface
interactions that would take place if the material were in the body and interacted with its surroundings.
Immobilization is an essential part of the SPR process and numerous scientific papers are dedicated to
the subject. The SPR chip is typically covered in a thin layer of gold. The substrate with thiol group at one
end can bind strongly to the gold surface via thiol-gold bond.
Each technique has several advantages and disadvantages. Ellipsometry is very similar to SPR in
that it utilizes a beam of light and measures the change of a property to determine the surface effects in
real time. However, ellipsometry only measures a very narrow range of light angles that are not always
optimal for protein adsorption. ELISA is able to determine the concentration of an absorbed substrate
but cannot determine real time kinetics of adsorption. Furthermore, ELISA requires the use of an
enzyme that can specifically target the substrate which is not always conveniently available. Circular
dichroism utilizes the changing polarization of light to test substances. CD is most effective at showing
changes in conformation. SPR is capable of determining real time kinetics and overall protein adsorption
amount. It is also very sensitive and can determine adsorption amounts as low as .1ng/ml [5]. However,
SPR also requires metal surface where the substrate can attach. Since peptides were designed with a
terminal sulfur atom that quickly and spontaneously bond to gold, this is not a problem for this
experiment. For these reasons, SPR was chosen as the method for determining protein adsorption.
Figure 1: Methyl Terminated SAM on Gold Surface
Figure 1 shows a common SAM surface. It is a methyl terminated SAM attached to a gold
surface.
Theoretical Background
The study described hereafter investigates the non-fouling properties of several protein chains.
Successful demonstration of non-fouling studies is one of the early steps in determining the
effectiveness of a protein as a drug carrier. The current drug carrier design includes three blocks or
sections: a hydrophobic section which lies on the inside of the sphere, a hydrophilic section which lies on
the outside of the sphere and resists fouling, and a ligand section which will be used for targeting
purposes. This study only focused on the hydrophilic section and tested the fouling resistance of several
amino acids. Four peptides were tested for their non-fouling capabilities. Figures 2, 3, 4, and 5 show the
structure of the peptides.
Peptide one, CV6, is a 7 amino acid peptide chain. The cysteine, C, amino acid provides the sulfur
which binds to the gold surface. V, valine, is a hydrophobic amino acid which gives the chain an overall
hydrophobic property. Peptide two, CV 6N3, is similar to the control peptide with three additional
residues, asparagine, N, on the end. Asparagine is a hydrophilic and uncharged amino acid with a
terminating amine. The addition of this peptide gives an overall hydrophilic surface quality. Peptide
three, CV4S2, includes serine, an uncharged polar peptide with a hydrophilic hydroxyl group. Peptide
four, CV4G2, has a glycine, G, in addition to the control peptide. Glycine is the smallest of all the amino
acids; the side chain is a hydrogen atom. This gives it a unique property where it is neither completely
hydrophobic nor hydrophilic but can exist in both environments. It is also uncharged.
CV6 does not fit all the criteria for a non-fouling surface since the surface is hydrophobic. But
since it is the backbone of all the other peptides, it serves as the control group. CV6N3 and CV4S2 fit both
criteria for a non-fouling surface. They have an overall neutral charge and hydrophilic properties... CV4G2
is neutral but does not as readily donate or accept hydrogen bonds and may or may not be considered
fully hydrophilic.
Figure 2: CV6 peptide
Figure 3: CV6N3 peptide
Figure 4: CV4G2 peptide
Figure 5: CV4S2 peptide
Table 1: Tested Peptide Properties
MW
Peptide Sequence (g/mol)
pI
Charge
CV6
715.94
5.5
-0.1
CV6N3
1058.25
5.5
-0.1
CV4G2
631.28
5.5
-0.1
CV4S2
691.83
5.5
-0.1
Experimental Procedure
In order to test for non-fouling of a surface, a self assembled mono layer needs to form on the
chip. The gold covered glass chip needs to be washed and treated with ozone to clean and activate the
surface. The chip is rinsed with pure ethanol, dried in an air stream, rinsed in de-ionized water, dried in
an air, rinsed with pure ethanol, and finally dried in an air stream. The chip is then placed in the o-zone
cleaner for thirty minutes. After, it is placed in the peptide solution with a concentration from 0.1 – 1
mg/ml for 24 to 48 hours to allow the SAM to form at room temperature. The SAMs form spontaneously
when the sulfur atom in cysteine binds with the gold surface. Peptides were purchased from NEO
peptide.
After SAM formation, the SAM surface was rinsed with water and then tested by SPR. In this
study, the surface was tested by the three most commonly studied proteins: Albumin, Lysozyme, and
Fibrinogen. These peptides were purchased from Sigma Aldrich. A 1 mg/ml solution is prepared for each
protein in degassed Phosphated Buffer Saline (PBS) also purchased from Sigma Aldrich. Before the
solution is created, PBS is degassed under vacuum for a minimum of twenty minutes to remove
dissolved air which limit the effectiveness and reduce accuracy of the SPR. Lastly, the chip is rinsed with
the same procedure as above.
The SPR chip is then placed on the crystal of the SPR and fixed in position. PBS is passed through
all of the sensing channels to provide a baseline as the software begins to collect and analyze the data.
PBS runs for approximately 30 minutes to continue the rinsing process and ensure only a monolayer of
surface remains. After approximately 30 minutes, all but one of the sensing channels is changed to the
dilute protein solution and run for approximately 30 minutes for the system to reach equilibrium.
Generally, non specific and specific adsorption kinetics are very fast and equilibrium is reached quickly.
Then, PBS again flows for approximately 10 minutes though the channels to wash away any unabsorbed
protein. The control channel accounts for any change in surroundings such as temperature. Any change
in the control, should also be seen in the other channels, so this effect can be removed.
The SPR sensor shows the refractive index of the solution and analyte on the chip at any given
time. After the baseline wavelength was found, this value was subtracted from all measured wavelength
to find the overall change in wavelength at any given time. The final Δλ value is the value used as a
measure of the protein adsorption ability.
Results
Two plots are presented below for each protein, and each plot has three characteristic phases.
The first section of the graph is a relatively linear horizontal section that represents the baseline for the
experiment. The second section is usually comprised of a quick jump in wavelength accompanied by a
slower increase. This portion is when the buffer solution being pumped over the chip changes to a
protein solution. The third section, a decay section with an asymptote, represents changing the protein
solution back to buffer and unabsorbed proteins washing away. The final change in wavelength is the
steady state value of Δλ at t=∞. This value is the value of Δλ at the end of the experiment.
Figure 6: SPR Data for CV6 Peptide
SPR wavelenght (nm)
λ vs Time for CV6
786.00
784.00
782.00
780.00
778.00
776.00
774.00
772.00
770.00
768.00
766.00
Fibrinogen
Lysozyme
PBS
Albumin
0
10
20
30
40
50
60
Time (min)
Figure 6 shows the results of the SPR on peptide CV 6. Figure 6 is the results of the original
experiment.
Figure 7: SPR Analysis for CV6 Peptide
SPR wavelenght (nm)
Δ λ vs Time for CV6
16
14
12
10
8
6
4
2
0
Fibrinogen
Albumin
Lysozyme
PBS
0
10
20
30
40
50
60
Time (min)
Figure 7 is the analysis of the change in wavelength caused by protein adsorption. Wavelength
shifts of approximately 5.2, 6.3, and 13.1 nm for albumin, lysozyme, and fibrinogen respectively were
found.
Figure 8: SPR Data for CV6N3 Peptide
λ (nm)
λ vs Time for CV6N3
756.00
754.00
752.00
750.00
748.00
746.00
744.00
742.00
740.00
Albumin
PBS
Lysozyme
0.00
50.00
100.00
150.00
Time (min)
Figure 8 shows the results of the SPR on peptide CV6N3. Figure 8 is the results of the original
experiment. Due to an error during experimentation, no usable fibrinogen data was obtained.
Figure 9: SPR Analysis for CV6N3 Peptide
Δλ (nm)
Δλ vs Time for CV6N3
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
Lysozyme
Albumin
PBS
0.00
50.00
100.00
150.00
Time (min)
Figure 9 is the analysis of the change in wavelength caused by protein adsorption. Wavelength
shifts of approximately 5.1,5.1 and 7.4 nm for albumin and lysozyme respectively were found. Due to an
error during experimentation, no usable fibrinogen data was obtained.
Figure 10: SPR Data for CV4G2 Peptide
λ vs Time for CV4G2
754.00
752.00
λ (nm)
750.00
748.00
Lysozyme
746.00
Fibrinogen
744.00
PBS
742.00
Albumin
740.00
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Time (min)
Figure 10 shows the results of the SPR on peptide CV4G2. Figure 10 is the results of the original
experiment.
Figure 11: SPR Analysis for CV4G2 Peptide
Δλ (nm)
Δλ vs Time for CV4G2
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
Lysozyme
Albumin
Fibrinogen
PBS
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Time (min)
Figure 11 is the analysis of the change in wavelength caused by protein adsorption. Wavelength
shifts of approximately 1.4, 3.1, and 5.9 nm for albumin, lysozyme, and fibrinogen respectively were
found.
Figure 12: SPR Data for CV4S2 Peptide
λ (nm)
λ vs Time for CV4S2
757.00
756.00
755.00
754.00
753.00
752.00
751.00
750.00
749.00
748.00
PBS
Albumin
Lysozyme
Fibrinogen
0.00
10.00
20.00
30.00
40.00
50.00
Time (min)
Figure 12 shows the results of the SPR on peptide CV4S2. Figure 8 is the results of the original
experiment.
Figure 13: SPR Analysis for CV4S2 Peptide
Δλ vs Time for CV4S2
7.00
6.00
Δλ (nm)
5.00
4.00
PBS
3.00
Fibrinogen
2.00
Albumin
1.00
Lysozyme
0.00
0.00
10.00
20.00
30.00
40.00
50.00
Time (min)
Figure 13 is the analysis of the change in wavelength caused by protein adsorption. Wavelength
shifts of approximately 0.5, 1.3, and 5.4 nm for albumin, lysozyme, and fibrinogen respectively were
found.
Discussion
Comparing the magnitude of the wavelength shift is comparable to comparing the effectiveness
of the non-fouling surface. As wavelength shift increases, protein adsorption increases. Table 2 compiles
the results.
For comparison purposes, we can compare these results to other common biomaterials that are
designed to resist protein adsorption. PEG, poly-ethylene glycol, is one of the commonly used
biomaterials in the world for non-fouling surfaces. Typical literature values for wavelength shifts are
between .5 and 1 nm [6]. We can also compare these values to a surface that not be considered nonfouling, such as a surface terminated with –CH3. Typical literature values for methyl terminated surfaces
are around 10 –15 nm [7]. Although these proteins do not resist protein adsorption as well as PEG, they
will most likely not require the same level of resistance. Furthermore, this study only details a few amino
acids and their affect on protein adsorption. Additional studies on single amino acids or groups of amino
acids may yield better results.
Table 2: A Comparison of Peptide Terminated Group and λ Shift
CV6
CV6N3
CV4G2
CV4S2
Fibrinogen
13.14
N/A
5.90
5.41
Lysozyme
6.33
4.99
3.11
1.33
Albumin
5.17
7.46
1.43
0.55
Table two shows that the effectiveness of the surface at resisting protein adsorption compared
to the control peptide. The addition of N, G, or S amino acids at the end of the control chain reduced
single protein adsorption in all cases except one (-VN and Albumin). Therefore, it is possible to conclude
that it is possible to reduce protein fouling. However, fibrinogen adsorption appears to be a problem for
all proteins tested. Although fibrinogen adsorption is less than common fouling surfaces, it is still
significantly higher than standard non-fouling surfaces. In order of effectiveness: -V4S2 > -V4G2 > -V6N3.
Currently, it is impossible to tell why there are subtle differences in wavelength shifts between
the peptides terminated in VN, VG, and VS. The asparagine and serine terminated peptides satisfy all
four requirements for non fouling surfaces. The most likely cause is the difference in how readily each
amino acid is to donate/accept hydrogen bonding, or the degree of hydrophilicity of each peptide.
Recommendations
A number of recommendations for further research can be made. First, to repeat experiments a
minimum of three times with fresh peptides to confirm repeatability. Second, to test additional peptide
termination groups to determine the most effective at resisting protein adsorption. Lastly, perform
contact angle measurement calculations to determine the degree of hydrophilicity of each protein to
help determine the cause of the different wavelength shifts.
References
[1]
Suhaas Aluri, Siti M. Janib and J. Andrew Mackay. Environmentally Responsive Peptides as
Anticancer Drug Carriers. Advanced Drug Delivery Reviews 2009; 11: 940-952.
[2]
B.D., Ratner,.. Biomaterials Science: An Introduction to Materials in Medicine. City: Elsevier
Academic Press, 2004.
[3]
Shenfu Chen, Lingyan Li, Chao Zhao and Jie Zheng. Surface Hydration: Principles and
Applications Toward Low-Fouling/Nonfouling Biomaterials. Polymer 2010; 23: 5283-5293.
[4]
Shengfu Chen, Lingyun Liu, Jian Zhou, and Shaoyi Jiang. Controlling Antibody Orientation on
Charged Self-Assembled Monolayers. Langmuir 2003, 19, 2859-2864.
[5]
Homola, Jiri. Surface Plasmon Resonance Sensors for Detection of Chemical and Biological
Species. Chem Rev. 2008 Feb;108; 108(2):462-93.
[6]
C. Rodriguez Emmenegger, E. Brynda, T. Riedel, Z. Sedlakova, M. Houska and A. Bologna Alles.
Interaction of Blood Plasma with Antifouling Surfaces. Langmuir, 2009, 25 (11), pp 6328–6333.
[7]
Lingyan Li, Shengfu Chen, and Shaoyi Jiang. Protein Adsorption on Alkanethiolate SelfAssembled Monolayers: Nanoscale Surface Structural and Chemical Effects. Langmuir 2003, 19,
2974-2982.