Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System Thesis by Dorothy Weichi Pan In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2016 (Defended May 5, 2016) ii  2016 Dorothy Weichi Pan ORCID: 0000-0003-4066-7750 All rights reserved. iii ACKNOWLEDGEMENTS There are numerous people to whom I am grateful who have made my thesis research possible and enriched my experience at Caltech. First of all, I would like to thank my advisor, Professor Mark E. Davis, for his mentorship during my PhD. He makes himself available to his students, especially when they have puzzling data and questions. For an MD/PhD student aspiring to bridge the space between the laboratory and the clinic, his wealth of experience in translational medicine, business, and entrepreneurship after having brought two therapeutics to clinical trials was a valuable resource. I’d also like to thank Professor Jim Heath, the chair of my committee, for his leadership with Caltech’s NanoSystems Biology Cancer Center, as well as my other committee members Professor Judy Campbell and Professor Dave Tirrell for their insights into proposing and planning projects. Executing a project that starts with chemical synthesis takes testing to the in vitro and all the way to the in vivo stage requires many instruments and facilities, as well as the expertise for a broad range of subjects. Dr. Yen Yun at City of Hope kindly provided pharmaceutical antibodies that I used as targeting agents for my project. I’d like to thank Dave Vandervelde in the liquid NMR facility for suggesting useful methods and aiding me in the analysis of my polymers. Mona Shahgholi in the mass spectrometry facility trained on a couple instruments that allowed me to obtain the masses of crucial material. I also worked extensively with Alasdair McDowell doing cryo-transmission electron microscopy to confirm the size of my nanoparticles. Andres Collazo trained me and helped troubleshoot problems with confocal microscopy. Suresh Gupta, the computer guru in the chemical engineering department, was able to save me a lot of time trying to fix the many iv computers operating instruments in our lab which crashed during my time performing experiments. The staff at the animal facility was also critical to performing mouse experiments, especially vet tech Gwen Williams for her thorough knowledge of procedures and intuition for mouse behavior, and veterinarian Dr. Karen Lencioni for her advice on caring for mice with tumors. Alyssa Maskell, Lorena Sandoval, and John Papsys provided animal husbandry for which I am appreciative. I would also like to thank the members of my lab for being wonderful colleagues. Han Han, Leonard Medrano, Yashodan Bhawe, and especially Devin Wiley and Jonathan Zuckerman, provided a lot of mentorship when I was getting my project started. Ben Boal worked in the chemistry hood across from me and was a source of advice in synthetic procedures. My MD/PhD colleague Andrew Clark, as well as Emily Wyatt and Dana Levine, are always willing to discuss ideas and experiments. I had the privilege of mentoring undergraduate Kristin Anderson for two summers, as well as Jan Winkler from ETH and Merle Bischoff from Aachen for their master’s theses, and I gained a lot of insight by helping them propose and guide them through their projects. Agnes Tong in the chemistry graduate program office was a valuable resource with all things related to requirements, paperwork, and much more. Martha Hepworth was also helpful with a lot of secretarial tasks. Roland Rapanot is a rock star administrator for the USC-Caltech MD/PhD program and facilitates the organization of the MD/PhD journal club where I first met my advisor Mark Davis and learned about the types of projects worked on in his lab. I’d also like to thank the former and current program directors Drs. Robert Chow and Steve Mittelman for their dedication to training and mentoring physician- scientists. v Outside the lab, I was very involved in many activities at Caltech. The Catalina Community Associates and the Resident Associates have been an amazing team to work with in building and fostering a residential community. I’d like to thank Felicia Hunt and Larissa Charnsangavej for their support of residential life, and my RA and CCA colleagues Beau Pritchett, Swarnima Manohar, Corey Reeves, Christine Morrison, Emily Wyatt, Daniel Brooks, Camille McAvoy, and many other CCA’s for their contributions, teamwork, and friendship. The Caltech Y is also a place that fosters a sense of adventure and service for the Caltech community. As a member of the outdoors committee, I’d like to thank the staff at the Y, Athena Castro, Portia Harris, Liz Jackman, and especially Greg Fletcher, for their support of all our student-led trips. Becky Schwantes, Jeremy Sandler, Casey Handmer, Howard Hui, William Frankland, Andrew Robbins, Joan Ballester, and Zoltan Tuza have been wonderful friends with whom I have led countless hikes and trips so that less experienced students could safely participate in adventures. Isaac Fees, Rebecca Rojansky, and I also led a Y hike where Rebecca (also an MD/PhD) and I had to deal with some altitude and medical issues that put our clinical reasoning to use in the wilderness! Having been a musician for about 80% of my life, I was glad to be able to play the flute and piccolo with the Caltech-Occidental Symphony Orchestra and in chamber music groups. The orchestra conductor Allen Gross chose a variety of major symphonic repertoire with flute and piccolo parts that were both fun and challenging for us to play. It was amazing to make music with a fantastic flutist and friend, Megan Newcombe, and getting a review from Delores Bing after a concert that the flute section is the best section in the orchestra. Megan and I were joined by Jeffrey Thompson, Jill Craven, and Jeremy Yager in the trombone section for dinner before orchestra rehearsal every week, and had vi conversations about music as well as careers – interviews, postdocs, and jobs – as they are all a step ahead of me in life after being a student. Concertmaster Sean Symon kept me company in another sound-proof practice room late at night in the music house. Zachary Erickson pestered me about the concerto competition until I finally decided to learn the Nielsen Flute Concerto and even got to play it with the orchestra. My flute teacher, Gary Woodward, is perhaps the most patient coach ever with an infinite number of ways to think about music and technical tricks up his sleeve. Hye-Sung Choe was a wonderful coach for my first chamber group at Caltech with Colin McKinney, Michael Zhang, and Jennifer Zhu playing Saint-Saens. Michael Kreiner has been a lot of fun to work with as bassoonist and coach for multiple of my chamber groups, including with Kelly Kim, Charles Cao, Jamie Rankin, and Aidan Chatwin-Davies. Aidan Chatwin-Davies was first a musical colleague, then a friend who also enjoyed hikes, but our relationship has become something more, and I appreciate him for supporting my hectic lifestyle. Lastly, I’d like to thank my family for telling me I was crazy to do an MD/PhD but letting me do it and supporting me along the way anyways. My sister, Tiffany Pan, started medical school at USC during my PhD and is now ahead of me in medical school, holding true to her assertion that she would never do an MD/PhD even when she had a chance to apply into the program. However, her stories about her sick patients when she comes home from the hospital reaffirms to me the need for more research and discovery especially in the fields of neurology and oncology, and I have much to look forward to as I head back into the clinical portion of my training. vii ABSTRACT Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective. Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP- PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection. Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug viii conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration. In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The ix nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo. Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment. x PUBLISHED CONTENT AND CONTRIBUTIONS Pan, D.W. and Davis, M.E. Cationic mucic acid polymer-based siRNA delivery systems. Bioconjugate Chem. 2015, 26, 1791-1803. doi : 10.1021/acs.bioconjchem.5b00324 D.W.P participated in the conception of the project, performed synthesis and experiments, prepared the data, and participated in the writing of the manuscript. Ahmed, M., Pan, D.W., and Davis, M.E. Lack of in vivo antibody dependent cellular cytotoxicity with antibody containing gold nanoparticles. Bioconjugate Chem. 2015, 26, 812-816. doi : 10.1021/acs.bioconjchem.5b00139 D.W.P participated in the conception of the project, performed experiments, prepared the data, and participated in the writing of the manuscript.