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Session A4 Paper # 91 Disclaimer—This paper partially fulfills a writing requirement for first year (freshman) engineering students at the University of Pittsburgh Swanson School of Engineering. This paper is a student, not a professional, paper. This paper is based on publicly available information and may not provide complete analyses of all relevant data. If this paper is used for any purpose other than these authors’ partial fulfillment of a writing requirement for first year (freshman) engineering students at the University of Pittsburgh Swanson School of Engineering, the user does so at his or her own risk. CAPSOSOMES AS A DRUG DELIVERY SYSTEM: A NEW WAY TO TREAT PHENYLKETONURIA Stephanie Thornton, [email protected], Vidic 2:00, Michael Zimlinghaus, [email protected], Mahboobin 10:00 Abstract—Capsosomes are an innovative medicine delivery method which allows for a more controlled and longer-term release of medication into the body. The structure of a capsosome consists of subunits containing the medication surrounded by a semipermeable membrane. This protects the drug molecules better than traditional pills so that they can reach their intended target site. The drug molecules suspended in the subunits of capsosomes are released more slowly into the body than if the drug were administered in traditional pill form. One application of this new technology is in the more effective and economical treatment of phenylketonuria (PKU). PKU is a metabolic disorder characterized by a deficiency in the enzyme necessary to break down the amino acid phenylalanine. The standard treatment for PKU involves regular doses of the enzyme that breaks down phenylalanine. This can be very costly, limiting the quality of care that is available to lower-income families. New developments in the field of drug delivery mechanisms, such as capsosomes, are showing promising results by delivering the necessary enzyme to the body more efficiently than traditional medication. Because of this, medication delivery via capsosomes will be required less frequently than with standard treatment, leading to less money spent by PKU patients. Once production efficiency is improved through more research and more widespread use, the cost of enzyme treatment via capsosomes will be substantially less. This development could change the way the world treats certain disorders, like PKU, and improve the lives of many people. and cofactors, which are used as the primary treatment for many metabolic and neurological disorders. While most pharmaceutical research is focused on identifying and testing new compounds for use as medications, comparatively little scientific work is being conducted for completely new drugdelivery systems. One of the most promising of the few methods that are being researched is capsosome-based medicine delivery. Capsosomes are microstructures built out of many small subunits consisting of drug molecules surrounded by a membrane made up of two polar layers of lipid molecules. Capsosomes are created by stacking the subunits one on top of the other in a spherical shell around a silica grain center, which acts as a foundation for the subunits. This construction technique is known as the layer-by-layer (LbL) technique. Capsosomes have been developed to distribute protein-based drugs to patients’ bodies more effectively by circumventing factors that would normally decrease the amount of the drug that reaches the target area. These factors include digestion and denaturation of the drugs by stomach acid, as well as the drugs binding to non-target receptor sites on body cells in the bloodstream. If they are proven to fix these problems, capsosomes will make the treatment of many diseases less expensive and more efficient. One disease which could utilize capsosomes for treatment is phenylketonuria (PKU), a rare metabolic disorder that leaves patients unable to digest most protein-containing foods. Because traditional treatment for this disease and others like it can be very expensive, patients and their families will benefit from a drug delivery system, such as capsosomes, that economizes the production and delivery of drugs that are already in use. Key Words—Capsosomes, Drug delivery systems, Enzyme Medication, Metabolic Disorder, Phenylketonuria, PKU Treatment INNOVATIVE MEDICINE THE STRUCTURE AND CHEMISTRY OF CAPSOSOMES The predominant ways for patients to receive drugs under modern medicine are either by taking pills orally or getting injections intravenously. While both methods have been proven effective at delivering relatively simple molecules such as those found in Aspirin, Benadryl, or Claritin, they are not nearly as effective at delivering proteinbased medications. Medications of this type include enzymes Both the structure and function of capsosomes are very intricate in nature. Their structure is perhaps best explained through an analysis of the processes used to construct them. Nano-biotechnology researchers led by Frank Caruso at the University of Melbourne worked on the development of this micro-container, and the construction is well detailed in their University of Pittsburgh Swanson School of Engineering Submission Date 3.03. 2017 1 Stephanie Thornton Michael Zimlinghaus journal published in Angewandte Chemie International Edition [1]. The procedure for constructing a capsosome begins with a microscopic silica grain as a catalyst. The silica grain is then covered with a polymer coating, known as a separation layer, that provides a secure surface on which the first layer of capsules can be anchored. These capsules, referred to as liposomes, each contain a small amount of medicine inside them. A typical capsosome will contain somewhere between three and six layers of liposomes depending on how high of a drug dosage is required and what kind of drug is being delivered. A separation layer is also placed in between each shell of liposomes to keep the layers intact and to maintain the structural integrity of the capsosome. At the end of the construction process, a final capping layer is added to cover the outermost layer of liposomes, and the silica grain center is removed [1]. LbL capsosome construction is emerging as the dominant method of capsosome production in the pharmaceutical industry. research conducted at University of Melbourne showed why these steps are necessary and optimize the functionality of the capsosomes. Chemistry of Precursor and Separation Layers Before the first layer of liposomes can be attached to the silica grain center, a covering made from steroid-based lipids must be implemented. This first layer, known as the precursor layer, is meant to provide a strong base layer to which the liposomes will adsorb, as well as allow the removal of the silica grain center at the end of the capsosome construction process. There exist multiple different chemical formulas for the remaining separation layers used in capsosome preparation. One of the most promising of these formulas, developed by C.Y. Yoo and his associate researchers at Seoul National University of Science and Technology, is a Velcrolike structure that alternates between negatively charged sodium hyaluronate (HA) chains and positively charged chitosan molecules, creating a checkerboard pattern of positive and negative charges [3]. In addition, smaller liposomes without any drug molecules inside are interspersed between the positively and negatively charged molecules on the separation layer. This formula is particularly useful because it creates a large amount of varied charge along the surface of the separation layer, which allows the polar heads of the liposomes being attached to bind strongly to the surface. The additional empty liposomes being added help contribute to this effect while also filling in empty gaps between the HA and chitosan molecules. Fewer gaps in the separation layer ensure that the layered liposomes stay intact while the capsosome is moved around the body. Since the capsosome will maintain its structure for a longer period with this chemical makeup, the drugs being delivered to the body will be released for a longer amount of time [3]. The Structure and Chemistry of Liposomes The structure of liposomes resembles that of a basic biological cell. They consist of a semipermeable lipid bilayer membrane surrounding a small amount of drug molecules. The outer membrane is called a lipid bilayer because it is made up of two layers of lipid molecules. Lipid molecules are characterized as having two “ends”; a hydrophilic, polar head and a hydrophobic, nonpolar tail. In a liposome, the hydrophobic tails of two lipid molecules move towards each other, forming a membrane that has hydrophilic heads on both the outside and the inside and hydrophobic tails in between them. The membrane is semipermeable because only certain compounds can pass through it. This is ideal for the function of a capsosome because the drug is protectively contained but what needs to exit the cell can leave. The structure of liposomes is conducive to drug delivery because it allows the drug molecules to stay intact inside the membrane until the entire liposome is broken down by the digestive system, ensuring that the drug stays inside long FIGURE 1 [2] Procedure for constructing a capsosome It might be difficult to understand the composition of a capsosome from just a description, but it makes more sense once observed in a diagram. The procedure stages for constructing a capsosome is well illustrated in figure 1, from an American Chemical Society article about capsosome research done in the Centre for Nanoscience and Nanotechnology at the University of Melbourne led by Rona Chandrawati [2]. The diagram displays the various steps that are involved in the creation of a capsosome, including the placement of a precursor layer, the placement of the alternating liposome and separation layers, the addition of the capping layer, and removal of the silica grain center. The 2 Stephanie Thornton Michael Zimlinghaus enough to be safely delivered into the bloodstream of the patient. Thus, having many liposomes contained within one structure will allow even more medicine to be delivered into the body, which is exactly what the scientists working with capsosomes are aiming for [1]. While taking too much or too little of an over the counter drug such as Zyrtec may not be of great consequence, patients ingesting drugs for diseases that require very specific dosages are at a greater risk of not receiving the correct amount of the drug. Protein-based medications are very susceptible to not reaching the bloodstream of the patient intact, resulting in improper dosage. A variety of factors can cause a discrepancy between the amount of drug ingested by the patient and the amount that effectively reaches the target area of the body. According to research on protein-based drug efficiency being conducted at the University of Melbourne, two of the most prominent factors are unintended digestion of the proteins in the stomach and incidental binding of the proteins to nontarget receptor sites in the bloodstream of the patient [4]. Both issues can be overcome if capsosomes are used instead of powders or pills. Since the drugs are contained within sturdy liposome compartments that are in turn anchored to the overall capsosome structure, the stomach cannot digest them as easily as traditional medications. Even if a protein-based drug were to make it through the stomach, there is still a risk that the protein or enzyme being delivered might accidentally bind to a receptor site on a cell that it is not supposed to, negating any effects the drug was intended to have. While capsosomes do not completely remove the risk of either of these two situations occurring, they do significantly reduce them. As efficiency is always a key factor when discussing drug-delivery systems, this makes capsosomes an important development in the field of medicine. Current Difficulties with Capsosome Construction Capsosomes are intricate microstructures that are the result of long, complicated, and tedious construction processes. Because of this, there are still a few outstanding problems that arise when they are being built. While these issues are not completely detrimental to the construction processes, they will need to be solved before capsosome production can reach an industrial-scale level of efficiency. When working on a scale as small as individual capsosomes, it is relatively common for the materials being introduced during construction to incidentally bond in unintended ways. For example, liposome clumping during production is a prevalent issue for researchers working with capsosomes. If even a small number of liposomes begin to clump during the LbL construction, the entire capsosome sample will be rendered unusable. Thus, researchers must take great care to follow the construction process very closely. The result of problems like liposome clumping is that capsosome sample production takes a long time to do correctly, especially for the level of purity that is required for precise research. Even if many highly-trained people were put to work constructing capsosome samples for industrial use in the same manner as current researchers, production would be incredibly inefficient and not economically viable at all. While researchers working with capsosomes can take time to carefully build capsosomes, the industrial-scale processes that will eventually be used to make capsosomes will be much more heavy-handed and time-constrained, while at the same time requiring more purity and fewer errors in the product. One of the largest challenges facing the industrialization of capsosome production today is how to marry the careful construction procedures that are currently in use with the increased economic viability and efficiency that come from the use of macro-scale industrial processes. Long-term Release Mechanism Capsosomes may provide a solution to this problem for patients that must take large, regular doses of medications. Instead of being absorbed into the body like a normal pillform drug, capsosomes are used up far less quickly. The human body takes a longer time to decompose capsosomes because of their highly intricate structure discussed above. Thus, an equivalent dose of a capsosome-enclosed drug will last longer than the same dosage given in pill form. The main factor that determines this difference is the fact that capsosomes must be dismantled liposome-by-liposome within the body. In addition, each individual liposome must be broken down, all of which requires more time and energy, increasing the longevity of the drug. In contrast, traditional pill-form drugs can be absorbed very quickly into the bloodstream because the raw chemicals are already separated from any restrictive structures. Thus, capsosomes that contain a higher number of liposomes will be more effective drug-delivery agents. Even though capsosome research is still relatively new, great strides have been made in increasing the efficiency with which liposomes are adsorbed onto each layer. Specifically, researchers at the University of Melbourne have been able to achieve 95% liposome adsorption efficiency. This means that, INNOVATION IN PROTEIN-BASED DRUG DELIVERY SYSTEMS The most common way of delivering protein-based medications into the body is by producing a powder-like form of the drug and molding into a pill, which is then taken in orally by the patient. While this is by far the dominant medicine delivery system in use today, it is not without its flaws. Taking in doses of medication in pill form requires that the pills be properly dosed to keep the level of the drug in the body optimized. Therefore, the patient must take pills multiple times per day to experience proper symptom relief, while being careful not to take too much and risk overdosing. 3 Stephanie Thornton Michael Zimlinghaus on average, 95% of the total space available for liposomes on each layer of capsosomes ends up being occupied by them. The researchers achieved this by utilizing cholesterolmodified PLL (PLLc) as an adsorption agent on the separation layers of the capsosomes. However, this efficiency level only lasted until the sixth layer of liposomes were deposited onto the capsosome. After this step, the researchers say, liposomes simply clump together without properly adsorbing onto the forming capsosome. This limit may still be overcome in the future if different compounds are used as adsorbing agents in the separation layers of capsosomes, but for the present it seems that this method will yield the most liposome-dense capsosome structures [2]. A team of researchers at the University of Melbourne led by James Maina published a study discussing the efficiency and release time of protein-based drugs delivered into the human body through capsosomes. The researchers found that capsosome usage greatly extends the period over which the protein-based drugs are released. They specifically compared the release percentage over 80 days of three different materials used to construct the liposomes [4]. combination had the highest overall drug release over the trial period, while the DPPC and DMPC/DPPC/DOPS combinations were both similar to each other, but significantly less than the DMPC/DPPC. One can see that all three of these compounds exhibited very high drug release activity over the first ten days, levelling off but still releasing significant amounts of the test drug over the remainder of the eighty days. These results are very promising for the future of capsosomes as a long-term drug delivery agent, showing that a relatively small amount of capsosome material can be used to give a large and long lasting dose of medication. For comparison, traditional drug-delivery methods will usually result in approximately 100% decomposition within ten days. From this, it is clear to see that capsosomes greatly extend the amount of time that a drug remains active in the patient’s body [2]. PHENYLKETONURIA: A METABOLIC DISORDER PKU is a rare, hereditary metabolic disorder characterized by the patient’s inability to digest the amino acid phenylalanine (PHE). This amino acid is present in nearly every food that contains protein, which makes it difficult to avoid. PKU is specifically caused by the absence of the gene encoding for phenylalanine hydroxylase (PAH), the enzyme that breaks down PHE in the body [5]. PAH typically converts PHE into the amino acid tyrosine in the presence of tetrahydrobiopterin (BH4), which is a natural substance found in the body. Fortunately, effective treatments have been developed for PKU that involve the patient ingesting artificial PAH and BH4, so PKU patients can live normal lives if they do their best to avoid high-protein foods and takes the appropriate dosages of medication. Specialized Diet The diet of a person with PKU includes restriction of many foods since PHE is so common. This limits the types of foods that can be safely eaten by people with PKU primarily to vegetables and fruits, starches such as potatoes, and mushrooms. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) lists that dairy products, meat, fish, chicken, eggs, and most beans and nuts should all be avoided by PKU patients as they may cause an unsafe spike of phenylalanine in their blood [6]. The sweetener aspartame found in many foods, drinks, medications, and vitamins needs to be avoided because it releases phenylalanine when digested. Following a specific diet can allow a person with PKU to live with less risk, but with this extensive list of restrictions, it can obviously be very challenging to deal with and they need additional treatment to stay healthy. PKU formula has been developed to provide the essential nutrients that people with PKU are not getting from their restricted diet. A newborn receives special infant FIGURE 2 [4] Cumulative liposome release percentage of three different liposome compound types The compounds used to make the materials are all derivatives of chemically similar lipid molecules, whose IUPAC names are as follows; 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), and 1,2-dioleoyl-sn-glycero-3phospho-L-serine (DOPS). The three compounds tested by the researchers were a combination of DMPC and DPPC, DPPC alone, and a combination of DMPC, DPPC, and DOPS [4]. The graph in figure 2 compares the cumulative liposome release percentage of three different liposome compound types over the course of eighty days [4]. The DMPC/DPPC 4 Stephanie Thornton Michael Zimlinghaus formula which can be mixed with breast milk or normal formula milk so that the child does not have too little or too much phenylalanine [6]. Older children and adults take a different formula which needs to be consumed every day. Additionally, health care professionals can recommend other supplements because each person with PKU can consume different amounts of phenylalanine. Symptoms and Statistics According to the HONCode certified Genetics Home Reference on the US National Library of Medicine website, if PKU is not properly identified and treated soon after a child is born, they can develop symptoms of hyperphenylalaninemia (H-PHE). These symptoms include jerky movement, seizures, developmental delays, behavioral problem, psychiatric disorders, and permanent intellectual disability [5]. Children born with PKU may appear normal at birth, but symptoms can appear after about three to six months. Untreated individuals could potentially be identified by having lighter skin and hair than unaffected family members and a strong ammonia-like body odor due to excess phenylalanine in the body [5]. Statistics provided by the NIH state that in the US, PKU occurs in 1 in 10,000 to 15,000 infants and most cases are detected during newborn screening tests [5]. This means treatment can be started immediately and the severe signs never show. Unfortunately, many countries without proper medical facilities are not able to properly identify PKU, and children who are afflicted with this disease are not able to receive proper treatment because of this. Even if PKU is identified, some families are not wealthy enough to afford the expensive treatments. Therefore, it is necessary for affordable treatments to be developed and improved. FIGURE 3 [7] PHE levels in person without PKU, person with PKU, and person with PKU being treated with KUVAN Figure 3 illustrates how the added KUVAN stimulates the PAH enzyme to work and process the PHE in the way it would process in a person without PKU. It also shows how a person with PKU that has not received treatment has the defective PAH enzyme which will not change. The image compares the process of breaking down PHE in a person without PKU, a person with PKU and no medication, and a person with PKU taking KUVAN to illustrate how the difference in PHE levels occurs. KUVAN can certainly help keep PHE levels low, but the NICHD describes how it is not a perfect solution to the problem because having too little BH4 is only one reason a person may not break down phenylalanine [6]. Therefore, it only helps some people and must be prescribed in addition to the typical PKU diet. Additionally, the FDA suggested that research continue for the KUVAN medication because long-term safety and effectiveness is still unknown. From this, it can be concluded that other treatment options still need to be developed. A lot of research has been conducted into discovering new treatments for PKU, such as a regularly injected dose of PAH or gene therapy to train the patient’s body to make PAH on its own. However, the journal from Advanced Functional Materials states how “gene therapy has only been employed with limited success due to poor efficiency of gene delivery into the liver and lack of sustained gene expression” [8]. Other potential treatments being researched and explored by scientists listed on the NICHD website include “large neutral amino acid supplementation”, which could help impede phenylalanine from entering and impairing the brain, and “enzyme replacement therapy,” which uses a substance that is TRADITIONAL PHENYLKETONURIA TREATMENTS As identified above, PKU is a rare disorder and requires large, regular doses of PAH. Because of this, treatment for this disorder can be expensive, and is often difficult for lowerincome families to afford. The first prescription medication for PKU approved by the US Food and Drug Administration (FDA) is the drug sapropterin dihydrochloride, called KUVAN, which is in the form of tablets or powder for oral solution. According to the official KUVAN website, the active ingredient is a pharmaceutical version of BH4, so taking KUVAN adds more BH4 to the body and stimulates the PAH enzyme to process PHE which converts it to tyrosine and lowers the level of PHE in the patient’s blood [7]. 5 Stephanie Thornton Michael Zimlinghaus much like PAH [6]. These treatment methods appear promising, but even if they are developed and made to be effective, they would still be quite expensive due to the rarity of the disease and the large amount of resources needed to produce these specialized medications. EXAMINING THE BIG PICTURE: THE FUTURE OF SUSTAINABLE CAPSOSOME PRODUCTION As of now, the processes used to construct the liposomes, separation layers, and the completed capsosomes are somewhat inefficient and hard to control. As with any innovation, these flaws will most likely be fixed with increased funding and research. The advantages that capsosomes hold over traditional pill-form drug delivery for protein-based medications will continue to grow with additional research being conducted on compounds used to create viable separation layers and liposomes. While capsosome production may soon become more efficient, it will likely still have a comparable overall cost to traditional pill-form drugs. However, the similar costs of production will yield different amounts of usable medication. Capsosomes provide a more efficient and controlled dosing mechanism than pills or powders, and thus allow for a higher dosage of the drug to reach its desired target within the body. This results in capsosomes being a more sustainable option for protein-based drug delivery. Although the cost of each dose may increase to a more due to a more complicated construction process, each dose will last longer, decreasing the number of doses needed in a given time frame to achieve the same effect. Once these production efficiency and accuracy roadblocks are overcome, capsosomes may become the primary drug-delivery method for all protein-based drugs due to the metabolic and economic advantages that they hold over traditional methods. Since capsosomes not only deliver these drugs more intact than traditional pill-form, but also increase the efficiency with which the body uses the drugs, the economic benefits of using capsosomes instead of pills will pay great dividends. The social sustainability aspect of capsosomes is highly favorable. If capsosomes become more commonplace as a drug-delivery mechanism, additional research positions will be created to study and improve production techniques and the chemical aspects of the construction process. Patients that require treatment through protein-based medications, which comprise a large portion of all people receiving medical treatment, will also benefit from capsosomes due to the increase in treatment quality that they will get. Patients that could potentially take capsosome-form drugs will benefit from further research because the price of drugs will decrease. Oftentimes, metabolic diseases like PKU require expensive and frequent doses of medication. A regimen such as this can put great financial strain on a family. If capsosome production becomes more economically viable, the overall cost of treatment for patients will decrease. Not only will this save money for people already receiving protein-based drugs, but it will also make treatment options available for patients in countries where widespread access to expensive pharmaceuticals is currently very restricted. CAPSOSOMES AS A TREATMENT MECHANISM FOR PKU Since other treatments are only proving to be mildly successful and PKU is causing a daily struggle in the lives of many people, it is necessary to investigate new drug delivery methods using innovative technology like capsosomes. A study conducted by researchers at the Aarhus University led by Leticia Hosta-Rigau and Brigitte Städler tested capsosomes loaded with phenylalanine ammonia lyase (PAL) to consider it as a potential method to treat PKU [8]. The goal was to test replacing the defective PAH enzyme with the PAL enzyme and protecting it from degrading too quickly in the digestive tract or bloodstream. The problem with many drug delivery methods with typical pills is that they degrade in the bloodstream before they reach the intended site of action and then they do not stay active for a long enough period. Additionally, enzymes taken orally in a regular pill or powder could be inactivated due to the harsh environment or the gastrointestinal (GI) tract. To treat PKU via capsosomes overcomes some early degrading issues because the advanced encapsulation structure of capsosomes protects the enzyme from unintended folding or denaturation. The PAL enzyme was selected by the researchers because the optimal pH of PAL is between 8.0 and 8.75 which is close to the average pH of the small intestine [8]. This enzyme is a non-human enzyme which can be obtained from plants, some fungi, yeast, and more. It converts PHE into the nontoxic trans-cinnamic acid (t-ca) which is then converted to benzoic acid in the liver and secreted via urine [8]. This means it will work to help PKU patients by lowering PHE levels in the body. The capsosomes are ideal because the polymeric carrier has a strong structural integrity, the liposomal compartments contain and protect the enzymes, and the semipermeable polymer membrane allows for the controlled release of the enzymes from the interior to the external environment in the body. The capsosome acts as an extracellular microreactor which enables the PAL enzymes to safely reach the intestine where they will then act to remove the unwanted PHE [8]. The research conducted demonstrated that the PAL capsosomes successfully conducted the enzymatic reaction to convert PHE to t-ca repeatedly in the presence of human intestinal epithelial cells while being exposed to a simulated environment of the intestine [8]. The results of their lab study with a dynamic environment like the intestine means they are one step closer to testing and possibly treating people with PKU via capsosomes containing therapeutic enzymes. 6 Stephanie Thornton Michael Zimlinghaus Treatment”. Advanced Functional Materials. Wiley-VCH Verlag. 07/01/2015. Accessed 01/24/2015. http://onlinelibrary.wiley.com/doi/10.1002/adfm.201404180/ abstract Spreading awareness of disease identification and proper treatment will follow the decrease in medication price, leading to more lives saved and financial burdens lessened across the world. Even though most financial gains to be had in the pharmaceutical industry still lie in the development of new medicinal compounds more funding should be allocated for the study of alternate drug-delivery systems, specifically capsosome drug delivery. Capsosomes are a critical development in this field because they can be used to treat a wide variety of diseases and disorders more effectively if their production efficiency is increased. Pills will still be widely used for quick, short term relief, but capsosomes are much more desirable if the proteins need to be delivered in large doses for long-term healing. They are the key to unlocking an entirely new way of treating metabolic diseases, and their prominence will lead to a decrease in suffering for many people around the world. ADDITIONAL SOURCES A. Balmer and P. Martin. “Synthetic Biology: Social and Ethical Challenges”. University of Nottingham Institute for Science and Society. 05/2008. Accessed 1/09/2017. http://www.synbiosafe.eu/uploads/pdf/synthetic_biology_so cial_ethical_challenges.pdf “Capsosomes’ To Treat Phenylketonuria.” Science and Technology Concentrates. 06.08.2015. Accessed 1.9.2016. http://rt4rf9qn2y.search.serialssolutions.com/?genre=article &title=Chemical%20%26%20Ent igineering%20News&atitle=%27CAPSOSOMES%27%20T O%20TREAT%20PHENYLKETONURIA. &author=J.%20K.&authors=J.%20K.&date=20150608&vol ume=93&issue=23&spage=24&issn=00092347 Helen K. Berry. (1979). USA Patent Number US4252822A. Accessed 1/09/2017. https://www.google.com/patents/US4252822 J. Kreuter et al. (1995). USA Patent Number WO1995022963A1. Accessed 1/09/2017. https://www.google.com/patents/WO1995022963A1?cl=en &dq=WO1995022963A1+patent&hl=en&sa=X&ved=0ahU KEwiJxdX_lL3RAhWC1CYKHRfkA5IQ6AEIGjAA SOURCES [1] F. Caruso. “A Microreactor with Thousands of Sub compartments: Enzyme-Loaded Liposomes within Polymer Capsules”. Angewandte Chemie International Edition. 2009. Accessed 1/09/2017. http://phys.org/news/2009-05-capsulesencapsulated-enzyme-equipped-liposomes-embedded.html [2] R. Chandrawati et al. “Engineering Advanced Capsosomes: Maximizing the Number of Sub compartments, Cargo Retention, and Temperature-Triggered Reaction”. University of Melbourne Center for Nanoscience and Nanotechnology. 03/01/2010. Accessed 01/09/2017. http://pubs.acs.org/doi/pdf/10.1021/nn901843j [3] C. Y. Yoo et al. “Preparation of novel capsosome with liposomal core by layer-by-Layer self-assembly of sodium hyaluronate and chitosan.” Colloids & Surfaces B: Biointerfaces. 08/2016. Accessed 01/24/2017. https://www.ncbi.nlm.nih.gov/pubmed/27085041 [4] J. Maina et al. “Capsosomes as Long-Term Delivery Vehicles for Protein Therapeutics” Researchgate.net. 07/2015. Accessed 1/09/2017. https://www.researchgate.net/publication/279942083_Capso somes_as_LongTerm_Delivery_Vehicles_for_Protein_Therapeutics [5] Genetics Home Reference. “Phenylketonuria” U.S. National Library of Medicine. 02/21/2017. Accessed 2/27/17. https://ghr.nlm.nih.gov/condition/phenylketonuria [6] “What are common treatments for phenylketonuria (PKU)?” Eunice Kennedy Shriver National Institute of Child Health and Human Development. Accessed 1.9.2016. https://www.nichd.nih.gov/health/topics/pku/conditioninfo/P ages/treatments.aspx [7] “PKU Basics.” KUVAN. 2017. Accessed 1.9.2016. http://www.kuvan.com/pku-basics/ [8] L. Hosta-Rigau et al. “Extracellular Microreactor for the Depletion of Phenylalanine Toward Phenylketonuria ACKNOWLEDGMENTS We would first like to sincerely thank our co-chair student advisor, Iman Basha, for being so kind and offering her assistance whenever we needed guidance. Additionally, we thank our writing instructor, Janine Carlock, for helpful comments and instruction through her analysis of our proposal, annotated bibliography, and outline. Thank you to Beth Bateman Newborg for providing us with the necessary directions to complete this assignment and her positive encouragement of everyone. Stephanie thanks her roommate, Kait DeOre for encouraging her to be productive. She also thanks Joshua Tarlo for keeping her company in the library while writing it. Michael thanks his best friend, Gary Xu, for being an inspiration to work hard. He also would like to thank a few of his other friends for providing examples of why it is a bad idea to procrastinate. Finally, we thank our parents for always supporting us and providing us with this amazing opportunity in the Swanson School of Engineering to learn applicable engineering and writing skills. 7 Stephanie Thornton Michael Zimlinghaus 8