Self-assembling Peptide Nanoparticles for Drug Delivery

Abstract

[evdate 2009-05-01] THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY President's Cup 2009 Final Report Self-assembling Peptide Nanoparticles for Drug Delivery CAI Bing a, CHOW Connie Hiu Ying b, LEUNG Man Wui c, YEUNG Chi Kin Jacky d a SID: Chemical and Biomolecular Engineering, Year 3, b SID : Chemical and Biomolecular Engineering, Year 4, C SID: Chemical and Biomolecular Engineering, Year 3, d SID: Chemical and Biomolecular Engineering, Year 4,

Endorsed by Dr. Ying, Chau, Department of Chemical and Biomolecular Engineering

Abstract As technology advances, so does the development of modern medicine. In the past decades, there has been an evolution in the way drug is delivered and administrated to patients. Insulin is no longer extracted from animals anymore but from genetically modified bacteria. Gliadel a brain cancer drug uses polymer for controlled drug release, has become the first newly approved treatment for brain cancer in 20 years. Such development of novel drug delivery does not only raise our standard of living but also created a billion dollar market in medical treatment. In our project, we investigated a new way of protecting drug molecules and delivering them to specific target sites in our body. By building a self-assembly triskelion nano molecule, (FPT)3--TAEA, we are able to create a vesicle in the tens of nanometers with the capability of encapsulating small drug molecules. Our results have shown that the vesicle is biocompatible and has the ability for specific drug release at designated location. Such technological break-through holds the promise of minimizing drug degradation and loss, preventing harmful side-effects and increasing the efficacy and efficiency in drug delivery.

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Table of Contents Abstract 1 Introduction 3 Prospective Market 4 Literature Review 5 Controlled Release Using Self-Assembling Nanofiber 5 The Natural Self-Assembling Protein - clathrin 5 Self-assembling Dipeptide Molecules 6 Study on the self-assembling drug, PLL 7 Bottom-up Design 8 Design Objectives 8 Design Specifications 9 Detail Description of Our Project 12 Synthesis of the dipeptide leg, Fmoc-Phe-Tyr-OH (FPT) 12 Conjugation of FPT and Tris(2-aminoethyl)amine (TAEA) 12 Discussion and Results 13 Synthesis of (FPT(tBu))3-TAEA 13 Synthesis of (FPT)3-TAEA 13 Identification of Vesicle Formation Conditions 13 Dye Encapsulation and Release by Diffusion 14 Negative Control - (FP)3-TAEA Vesicle Formation 16 Future Work 16 Conclusion 16 Appendix 17 References 19

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Introduction For the past decades, technology in drug delivery has evolved at a lighting speed. From the ancient practice of applying ointments on skin to the modern chemotherapy used in cancer therapy, engineers are constantly looking for ways to invent novel drug delivery method which could specifically act on a target site. However, this is extremely difficult as the body is designed with transportation systems that are effective in carrying nutrients, and hence with highly toxic drug along throughout the body. This means that highly toxic drugs that are designed to kill cancer cells would be circulating around the body, causing damage, pain and other side effects to the rest of the body.

To overcome this problem, the incorporation of targeted controlled release strategies into the design of drug delivery systems becomes extremely important. This system will need to be efficient, biocompatible, robust and useful for diverse applications so that it can deliver active compounds to the targeted sites. Two complementary strategies can be used for the design of such a system. The 'top-down' approach strips down complex entities into smaller components, while the 'bottom-up' approach assembles the novel supramolecular architecture molecule by molecule.2

In this project, our team has adopted the 'bottom-up' design approach and has investigated a novel method in target drug delivery. Through building a triskelion dipeptide molecule with self-assembling ability and enzyme sensitivity, our team has discovered the potential of drug encapsulation and targeted delivery through the assembly and disassembly of this molecule. Our results have shown that the molecule would spontaneously self-assemble into vesicles with a hollow interior for drug encapsulation. In the presence of kinase, an enzyme that is found abundantly in cancer cells, these vesicles would disassemble and release chemotherapic drug at the designated location.

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Prospective Market With the development of new drug delivery technology, the pharmaceutical industry has poured in billions of dollars into the development of new methods for targeted drug delivery. With the increase in investment from corporations, the sales of advanced drug delivery systems were over US$54.2 billion in 2004 in the United States alone. In 2005, it reached US$64.1 billion. This market will continue to grow at an average annual growth rate (AAGR) of 15.6% and will reach US$153.5 billion by 2011.3 The annual sales of some drugs using advanced drug delivery systems are listed below4 : Product (Company) Product description (usage) Annual sales (USD) Procrit(Amgen/J&J) Erythropoietin (Treatment of anemia) 2.7 Billion Epogen (Amgen) Erythropoietin (Treatment of anemia) 2.0 Billion Intron A (Schering Plough) Interferon-a (Treatment of leukaemia) 1.4 Billion Enbrel (Immunex) Monoclonal antibody (Treatment of rheumatoid arthritis) 700 Million Cerezyme (Genzyme) Glucocerebrosidase (Treatment of Gaucher's disease) 500 Million Herceptin (Genentech) Humanized monoclonal antibody (Treatment of breast cancer) 500 Million Recombinant Insulin (Various Companies) Insulin (Treatment of diabetes) 3.4 Billion 2.1 Billion 1.1 Billion Table 1 - Annual sales of drugs using various drug delivery methods in the United States

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Literature Review To understand the development of this industry, our research has highlighted three different literatures which provide insights to our project.

Controlled Release Using Self-Assembling Nanofiber5 The first literature discusses about small peptides with self-assembling ability. The small peptides were used to form hydrogel for drug delivery purposes. The research shows that ionic self-complementary peptides with 16 amino acids (RADA16) can self-assemble into hydrogels with 99.5% water. Fluorescent dye was added into the hydrogel to determine of its release profile. The experimental results show that peptide with self-assembling ability can be used to trap small molecules and hence encapsulate drug molecules.

The Natural Self-Assembling Protein - clathrin The second literature introduces a natural self assembling protein - clathrin. Clathrin is a cytoplasmic protein located on the inner cell membrane, serving as an assistant in intracellular transportation mechanism for cells. A clathrin molecule takes the form of a triskelion, a figure with three similarly bended legs springing from a common center. 6 Under slightly acidic conditions, clathrin triskelions have the ability to spontaneously assemble into a cage by interacting with other triskelions, forming a supportive network around the enclosing cell membrane. Clathrin triskelions are able to form cages of different sizes, giving flexibility in sizes of cell membrane vesicles.7 Having a triskelion structure allows the molecule to form a structural sound vesicle without much energy consumption. CARGO areceptor vesicle formation

Figure 1 - Structure of clathrin triskelion, a cage formed by clathrin triskelions and mechanism of clathrin cage formation.

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Self-assembling Dipeptide Molecules The third literature discusses a dipeptide molecule with self assembling property. Previous research has shown that dipeptide, molecule with two amino acids, has the ability to self assemble into complex structure. This research paper shows that a triskelion structure using TAEA as the core center with 3 dipeptides (Tryptophan-Tryptophan) attached to each leg of the triskelion forms self-assembled structures. Using high-resolution transmission electron microscopy (HR-TEM), the team observed vesicles with distinct bilayer-like boundary with diameter of larger than 200nm. The research also concludes the self assemble nature is contributed by the IT-IT stacking between the aromatic rings on the dipeptides. The condition for the formation of vesicles is observed in 60% and 90% methanol solution after 7 days.8 Figure 2 - Triskelion structure designed by previous research teams. d) I) Figure 3 - Electron microscopy analysis of the self-assembled structures formed by the triskelion ditryptophan conjugate.

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Study on the self-assembling drug, PLL With the knowledge based on literature research, our team investigated the possibility of self-assembly using the drug molecule itself. Fmoc-Leucine is a common drug used for the treatment of inflammation. Based on the understanding of self assembly ability shown previously, experiments were set up using three amino acids, namely Fmoc-Leucine (Fmoc-Leu), Fmoc-Phenylalanine (Fmoc-Phe), and Fmoc-Lysine (Fmoc-Lys) to see if controlled drug release is possible in the form of hydrogel using the drug molecule, Fmoc-Leucine itself.

Our team used Fmoc-Phe, Fmoc-Lys and Fmoc-Leu (abbreviated as PLL) to form a self-assemble structure. An equal molar amount of amino acids were added to pure water and placed in a 60°C water bath until all dissolved. The solution was then left in room temperature overnight.

Our experimental result shows that the solution solidified and formed a gel-like structure. This gel-like structure is the result of the self-assembly among the three amino acids. Our experimental result also shows that after the gel is formed, Fmoc-Leu starts to leak out of the gel and dissociates into the medium. The graph below shows the release profile of Fmoc-Leu across time. This slow release can potentially be used as a controlled release in drug delivery. PLL release profile in nano pure water

Figure 4 - Fmoc-Leu release profile in PLL experiment

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Figure 5 - (i) Freshly made PLL gel in Phosphate Buffered Saline (PBS). (ii) PLL gel in nano pure water after two day. (the release of Fmoc-Leu is illustrated by the milkiness of the upper medium) (iii) Clear PLL solution after incubation in 60C hot water bath. (iv) PLL gel formed in room temperature.

Although the experimental result proves that drug molecules can be used directly in controlled drug release, such drug delivery method posts a major disadvantage: it can only be used if the drug itself has self-assembling ability. It cannot be applied to drugs that do not have such capability. A better controlled drug release system should be able to encapsulate drug molecules and transport the drug molecules. Hence our team develops a self assemble nano-scale vesicle that can physically encapsulate drug molecules for transportation in the body.

Bottom-up Design The self-assembling drug carrying vehicle (FPT)3-TAEA -

Design Objectives With the literature review and the formation of gel-like PLL in mind, our team develops four key critical objectives for our drug delivery system. We would like to synthesize a drug delivery material by mimicking the clathrin triskelion structure with the following characteristics.

First, the molecule needs to be biocompatible. Water solubility is one of the essential requirements for biocompatibility. In previous research, the condition for vesicle formation requires the use of methanol, which is highly toxic to human body. Using

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such vesicles in drug delivery would not be favorable under such condition. Second, the molecule needs to have the ability to self-assemble into a vesicle. Having this property allows the molecule to encapsulate drug spontaneously. The reason is that self assembly of molecules leads to a lower Gibbs free energy, thus making it more thermodynamically favorable as assembled molecules. Stability of the vesicle can be achieved due to this energetically favorable reaction, allowing drug-carrying vesicles to stay longer inside the body and hence reducing the dosage needed.

Third, the molecule has to be small and has the capability of forming vesicle in the tens of nanometer. Having such a small size reduces the chances of nano-size drug molecules leaking out of the cage-like structure uncontrollably through the pores by diffusion. In this way, it can reduce the potential disadvantages post by drugs, including toxicity, pain and undesirable side-effects.

Having such a small size is also essential for drug to retain longer inside the body. With particle size less than 60nm, the clearance half-life by the body is between 1-16 hours. Anything larger than 60nm could dramatically reduce the half-life. Also, the small size helps vesicles to penetrate through capillaries and release its content to the deepest part of the body. If the molecule is large, endocytosis is not possible and the cell membrane might block the entry of the vesicle into the cell, hence reducing the efficacy of the drug.

Last but not least, the vesicle needs to be sensitive to specific enzymes. In the presence of this enzyme, disassembly will be triggered readily to deliver drugs. Having this enzyme specificity allows the drug to be, and only be delivered to targeted cells where the specific enzyme is present. Having this targeting method allows the drug carrier to release drug selectively.

Design Specifications

With the above four objectives, our team designed the molecule with a triskelion structure. The self-assembling molecule consists of two main components. First is a three-legged structure known as Tris(2-aminoethyl)amine (TAEA) which serves as the core centre for the designed molecule. The second part is a dipeptide, Fmoc-Phenylalanine-Tyrosine (FPT), formed by amino acid Phenylalanine and amino acid Tyrosine. The N-terminus of Phenylalanine is protected by 9-fluorenylmethoxycarbonyl (Fmoc) while the C-terminus of the Tyrosine is covalently

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joined to one of the legs of TAEA. With one TAEA serves as the cores centre, three dipeptide molecules are covalently bonded to each of the three legs of TAEA respectively, forming a triskelion structure. TAEA 3 FPT didpeptide legs

Figure 6· Formation of triskelion molecule, (FPT)3-TAEA

The self-assembling property of the dipeptide molecule arises from non-covalent bonding, which can be cleaved relatively easily for disassembly. Non-covalent hydrogen bonds are formed by the interaction between hydroxyl groups from Tyrosine residues and carboxyl groups from other dipeptide molecules. The aromatic side chains of the dipeptide and the fluorenyl groups of Fmoc form π-π stacks by the overlapping of electron shell with each other. When π-π stacks and hydrogen bonds are formed, the heat released compensates the decrease in lithe disorder of the system', entropy.

Having a lower energy state in the assembled form makes it more favorable for molecules to group together. This is the reason why FPT can self-assemble without extra energy. Other components also contribute to the formation of vesicle and Table 2 summarizes the properties and functions of each component in the did peptide molecule, (FPT)3-TAEA.

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Abbreviations Full name Structure Specification Tris(2-aminoethyl)amine r NH2 1. Provide a core three leg structure TAEA 1. Protecting the %0 N-terminus of Phe Fmoc 9-fluorenylmeth oxycarbonyl 2. Fluorene group provides π-π stacking for self-assembling Phe Phenylalanine 1. Aromatic group helps self-assembling Tyr Tyrosine 1. Hydrophilic property 2. Aromatic group helps self-assembling phosphate phosphate 1. Enzyme-responsive II 2. Hydrophilic property

Table 2 - Structure, properties and functions of the S different components in the did peptide molecule, (FPT)3-TAEA & (FPTP)3-TAEA

The designed molecule, (FPT)3-TAEA is the primary unit for forming nano-scale vesicles which has hollow centers for drug encapsulation. Enzyme specificity is achieved by the interaction between the dipeptide molecule with a phosphate group (P). When a phosphate group comes close with FPT in the presence of an enzyme known as kinase, it will react with the (-terminus of Tyrosine and forms (FPTP)3-TAEA. Phosphate, being a negatively charged group, would repel each other. This repelling force provides the energy needed to overcome the thermodynamic stability of the self assembled vesicles and disrupt stability of the entire structure of the vesicle, breaking it back to the unassembled units, (FPTP)3-TAEA. The reaction by kinase is illustrated in the figure below.

Self-assemble into a vesicle Diassemble

Figure 7 - Assembly and Disassembly of molecule, (FPT)3-TAEA & (FPTP)3-TAEA

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Detail Description of Our Project With the working mechanism and design in mind, the next step is to synthesize the designed molecule, (FPT)3-TAEA. The synthesis is divided into two stages. First is the synthesis of the dipeptide legs, FPT. Second is of the conjugation of FPT with the triskelion center, TAEA.

Synthesis of the dipeptide leg, Fmoc-Phe-Tyr-OH (FPT) The synthesis of FPT follows a conventional peptide coupling procedure. The starting materials are Fmoc-Phe and Tyrosine(tert-Butyl) (abbreviated as Tyr(tBu)). As the carboxyl group in Fmoc-Phe resists binding with Tyr(tBu), an activation step is needed before coupling to increase the yield.

This activation replaces the hydroxyl group of Fmoc-Phe with a more bulky leaving group, Dee (N,N'-Dicyclohexylcarbodiimide). The replacement of the negatively charged hydroxyl group enhances the electrophilicity of Phe, hence enhanced the coupling reaction.9 Sodium bicarbonate is added to increase Tyr(tBu) polarity, hence allowing it to dissolve better in the medium and facilitate the reaction. The activated Fmoc-Phe is then allowed to react with Tyr(tBu) overnight to form FPT(tBu) . After the coupling reaction, FPT(tBu) is purified by precipitation using Diethyl Ether. The tBu group will later be removed by the addition of a strong acid, Trifluoroacetic acid (TFA), forming FPT. The resulting products are then purified by reverse-phase high pressure liquid chromatography.

Conjugation of FPT and Tris(2-aminoethyl)amine (TAEA) After purifying the did peptide legs, FPT, they are allowed to conjugation with the three legged center TAEA. The starting materials are pure FPT and TAEA, where FPT is added 5 times in excess to ensure a complete reaction with TAEA. The carboxyl group of FPT is first activated by HOBt and BOP, enhancing the reactivity of the molecule. An organic base, DIPEA is added to provide an alkaline condition to prevent NH2 from forming NH3+ which has a lower reactivity. After activating the carboxyl group, FPT is allowed to react with TAEA overnight to form (FPT)3-TAEA.

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Discussion and Results During the synthesis phase, our team has encountered several problems. These problems have affected our progress of synthesizing the designed triskelion. Below describes some of the problems we have encountered and how our team solved these issues. After synthesizing pure (FPT)3-TAEA, the methods of characterizing the vesicles and their capability of drug encapsulation will also be discussed in the later session.

Synthesis of (FPT(tBu))3-TAEA In the beginning of the project, the Tyrosine on FPT was thought to be too reactive due to the presence of a carboxyl group on it. The relatively high reactivity of the carboxyl group may reactive with other reagent during the synthesis process and affect the yield of the final triskelion molecule. Knowing this, our team decided to synthesize the triskelion molecule, (FPT(tBu))3-TAEA instead. The tBu would act as a protective group to the carboxyl group, preventing it from forming side products. With this (FPT(tBu))3-TAEA, tBu would then be removed to trigger self assembly. FPT(tBu) was synthesized successfully (Appendix I). However, FPT(tBuL could not be attached to the TAEA center successfully. Evidence had shown that there were presence of incomplete reactions, forming (FPT(tBu))-TAEA and (FPT(tBu))2TAEA. (FPT(tBu)3-TAEA, however, were rarely found (Appendix II). Steric hindrance is a possible reason, meaning that there was insufficient space for a third FPT(tBu) to attach to the TAEA core as tBu is a bulky group.

Synthesis of (FPT)3-T AEA Although tBu provides protection to Tyrosine, it also prevents the formation of triskelion with TAEA. Therefore, our team decided to remove the protective group before the conjugation with TAEA to reduce the effect of steric hindrance. Hence, FPT was used directly to conjugate with TAEA to form the triskelion. The results showed (FPT)3-TAEA was successfully synthesized (Appendix III).

Identification of Vesicle Formation Conditions Once the triskelion molecule is synthesized, conditions for forming vesicle for drug delivery purposes are investigated. It is expected that the feasibility of vesicle 13

formation and vesicle size will vary in different conditions, for example the solvent use, temperature and pH. A study on optimal vesicle formation condition is carried out to determine a suitable environment for vesicle formation.

Purified TAEA-(FPT)3 was saturated in three different conditions that are close to the human body environment. The three conditions were solvents sodium bicarbonate solution (pH 7.4), phosphate buffered saline (PBS), and nano-pure water. These samples were then incubated under room temperature for self-assembly. The presence of vesicles was then determined by two methods, Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM).

Figure 8 - Transmission Electron Microscopy analysis of the designed self-assemble vesicles in Sodium Bicarbonate Solution (pH7.4)

Results were positive and aligned with the design of our team. Both TEM and DLS results showed the presence of nano size vesicles with diameter between 20-40nm in Sodium Bicarbonate solution. In the other two solutions, although no vesicles were identified, nano-particles of different sizes were found. Due to the presence of nano-sized particles, it is believed that vesicles can also be formed in PBS and water with suitable alteration of the incubation conditions (e.g. sample concentration and temperature). Further investigations will be conducted in the future.

Dye Encapsulation and Release by Diffusion Once the vesicle is formed, our team investigated its ability to encapsulate drugs. To start with, sodium fluorescein, a dye employed clinically for angiography of the retina10

is used for drug release study. Sodium fluorescein is a self-quenching dye that if it remains inside the vesicle, its luminosity is reduced.

14 (FPT)3-TAEA was cultivated under dye containing sodium bicarbonate solution for vesicle formation. Dye that was not encapsulated was washed thoroughly by dialysis before further analysis. Under TEM, it is observed that the previous hollow structure is now filled with dye (darker in color).

Figure 9 - Transmission Electron Microscopy analysis of drug encapsulation capability of (FPT)3-TAEA vesicles. (FPT)3-TAEA vesicles has a cage-like structure with pores on it. When the dye leaks out through these pores by diffusion, fluorometer can be used to detect its luminosity and thus determines the concentration of drug release out of the vesicles. With the information, a release profile of the vesicle by diffusion can be produced. Below shows a preliminary release profile by diffusion for 5 hours. The slow release of dye indicated the possibility of controlled drug release a dye will not leak out fast from the vesicle pores. Time (min)

Figure 10 - Release of sodium fluorescein by diffusion

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Negative Control- (FP)3-TAEA Vesicle Formation The purpose for a negative control experiment is to investigate the necessity of using dipeptide versus one single peptide in the formation of vesicle. Using a single peptide molecule would eliminate the needs of forming the dipeptide, FPT. Fmoc-Phe can be used directly to react with TAEA, forming (FP)3-TAEA. It also provides an insight on the minimum number of amino acid needed for vesicle formation. Fmoc-Phe was used to react with TAEA and the result shows no vesicle formation with one amino acid. This shows that (FPT)3-TAEA is the essential basic unit for our self-assembling structure.

Future Work Once the release profile by diffusion is completed and the optimal vesicle formation condition is identified, our team aims to further investigate the release profile under different conditions and thus to identify what types of drugs would be suitable for encapsulation in the future: 1. The effect of temperature 2. The effect of pH 3. The effect of mechanical strength 4. The size of drug being encapsulated 5. The presence of enzyme, kinase, and phosphate groups 6. The presence of enzymes other than kinase

Conclusion

Our experiments have shown promising results of using dipeptide molecule with triskelion structure in drug transport and release application. We also showed the possibility of vesicle formation by (FPT)3-TAEA in biocompatible environment, hence improving the probability of applying this technology in modern medicine. Overall, we have achieved the four designed specifications stated at the beginning of the project (Nano scale, self assembly, enzyme sensitive and biocompatible). This project has shown significant results and further work would be needed to determine the efficacy and efficiency of using such method.

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Appendix Molecules to be are first ionized by cation with Molecular Weight (MW) of 1 mg/mmol, 23 mg/mmol, and 39 mg/mmol. When the newly charged molecules are introduced into an electric and/or magnetic field, their paths through the field are a function of their mass-to-charge ratio, m/z. the results will then be displayed over the mass spectrum (the x-axis). In short, if one wants to identify a molecule with MW = 100 mg/mmol, one needs to look for peaks at 101 (100+1),123 (100+23) and 139 (100+39). These respective peaks all represent molecules with a mass of 100mg/mmol. MW of FPT(tBu) = 606.8mg/mmol Appendix I - Mass Spectrum of crude FPT(tBu) product

17 Appendix II - Mass Spectrum of crude (FPT(tBu)3-TAEA product Appendix III - Mass Spectrum of crude (FPT)3-TAEA product 18 References 1 Gliadel Press Release, 'Implantable wafer is first treatment to delivery chemotherapy directly to tumour site' 2 S. Zhang, 'Fabrication of novel biomaterials through molecular self-assembly', Nat. Biotechnol. 21(2003) 1171-1178. 3 BCC Research, 'Advanced Drug Delivery Systems: New Development, New Technologies' (2006), Pub ID: WA1398775 4 Lecture Notes, CENG366, 2007 Spring Semester, Department of Chemical and Biomolecular Engineering, Hong Kong University of Science and Technology 5 Yusuke Nagai, Larry D. Unsworth, Sortirios Koutsopoulos, Shuguang Zhang, 'Slow release of molecules in self-assembling peptide nanofiber scaffold', Journal of Controlled Release 115(2006) 18-25 6 Joel A. Ybe, Frances M. Brodsky, Kay Hofmann, Kai Lin, Shu-Hui Liu, Lin Chen, Thomas N. Earnest, Robert J. Fletterick, Peter K. Hwang, 'Clathrin self-assembly is mediated by a tandemly repeated superhelix', Nature 399(1999) 371-375 7 Anna Young, 'Structural insights into the clathrin coat', Seminars in Cell & Developmental Biology 18(2007) 448-458 8 Surajit Chost, Meital Reches, Ehud Gazit, Sandeep Verma, 'Bioinspired Design of Nanocages by Self-Assembling Triskelion Peptide Elements', Angewandte Chemie Int. Ed. 46(2007) 2002-2004 9 Exploring Proteins, Reading Materials, BISC103, 2005 Fall Semester, Department of Biochemistry, Hong Kong University of Science and Technology 10 Michael R. Niesman, Bahram Khoobehi, Gholam A. Peyman, 'Encapsulation of Sdium Fluorescein for Dye Release Studies', Investigative Ophthalmology & Visual Science 33 (1992) 2113-2119 19