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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.