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Project Report Winter 2016


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Project Report Winter 2016

  1. 1. Theresa Rizk Mooney Lab for Cell and Tissue Engineering PI: Dr. David Mooney Mentor: Theresa Raimondo Exploring the Role of Phagocytosis in Nanoparticle Directed Macrophage Polarization Introduction A current medical challenge involves the complications of chronic inflammation, which can contribute to many inflammatory diseases including atherosclerosis, asthma, rheumatoid arthritis, and even cancer [1]. Acute inflammation is a necessary, protective response that kills invading pathogens, which could otherwise lead to disease and progressive tissue destruction. Uncontrolled activation of immune cells however, specifically macrophages, leads to chronic inflammation and can result in tissue damage. Classically-activated macrophages (M1) promote inflammation through the release of several proinflammatory signals, while alternatively- activated macrophages (M2) serve a regulatory function as anti-inflammatory cells (M2c), and play a key role in tissue healing (M2a). Initial research by my mentor suggests that IL-4 conjugated gold nanoparticles promote macrophage polarization towards the M2a phenotype in vivo, in an animal model of ischemic inflammation. This results in improved functional recovery of muscle strength following ischemic injury. The aim of my research was to study the mechanism by which the nanoparticles direct macrophage polarization; more specifically, to study the role of macrophage phagocytosis in nanoparticle-directed polarization. These results will be used to direct the next iterations of nanoparticle design. ResearchSummary Three drugs are used extensively in the literature to inhibit phagocytosis by different mechanisms: Chlorpromazine, an inhibitor of clahtrin-dependant endocytosis; Nystatin, an inhibitor of caveolae-dependent endocytosis; and Cytochalasin D, an inhibitor of phagocytosis/macropinocytosis [2]. Initial attempts at comparing the effects of these three drugs
  2. 2. Theresa Rizk Mooney Lab for Cell and Tissue Engineering PI: Dr. David Mooney Mentor: Theresa Raimondo indicated cytotoxicity, despite using dosages reported in the literature. The increased cytotoxicity likely resulted from the increased duration of treatment (3d), as compared to that reported in the literature (<3hrs); this longer duration was required to observe macrophage polarization and better represent the in vivo process. This challenge was addressed by making a concentration curve for each compound, in order to determine the appropriate concentration of each. The results allowed for choosing adjusted drug concentrations for future experiments which minimized cell toxicity. The most recent experiment was then designed to compare the effects of IL4-conjugated nanoparticles on macrophage polarization with and without phagocytosis inhibition. This was accomplished by establishing two main experimental groups: cells exposed to free IL4 in solution, and cells exposed to IL4-conjugated nanoparticles. Each main subset contained a positive control without inhibiting drugs, in which phagocytosis could occur freely, and experimental groups with each of the three phagocytosis inhibiting drugs, along with a negative control group in which cells were not exposed to IL4 at all. The experiment was run successfully, and several unexpected but informative results were observed. Some were due to unanticipated interference from the drugs, which continued to reduce viability and interfered with polarization. Cytochalasin D and Nystatin continued to reduce viability at the adjusted doses; interestingly however, the viability was improved more significantly following treatment with IL-4 conjugated particles as opposed to soluble IL4. It was also observed that Chlorpromazine was not effective at inhibiting phagocytosis at the concentration required to minimize cytoxicity, indicated by a pink cell pellet in the condition containing Chlorpromazine and the red nanoparticles. Reduced macrophage polarization following treatment with either Cytochalasin D or Nystatin in the presence of IL4 conjugated nanoparticles or soluble IL4 suggests that
  3. 3. Theresa Rizk Mooney Lab for Cell and Tissue Engineering PI: Dr. David Mooney Mentor: Theresa Raimondo membrane caveolae and actin polymerization are required for IL4 driven M2a macrophage polarization. The next priority in the ongoing research is to determine a more effective method of controlling phagocytosis, either by adjusting the design of the nanoparticles, or conjugating the IL4 to the surface of the well plate, in order to avoid the unwanted side effects of the drugs we’ve experimented with thus far. This will allow progress with cleanly examining the effects of phagocytosis on polarization. Reflection My research project thus far has proven to be a wonderful learning experience in research, experimental design, and overcoming challenges and obstacles. It has been one of my most involved experiences in terms of problem solving and thinking through complicated results, as well as a unique opportunity to be part of a project from its beginning. I have enjoyed the opportunity to guide the project. I have also greatly expanded my knowledge and awareness of chronic inflammation, it’s implications and solutions, and am continuously more fascinated by its medical consequences. Mentors My faculty mentor, Dr. Mooney, has been kept informed of my research endeavors and has been very supportive. I plan to present the comprehensive results from the past semester to him soon after the winter holidays. My lab mentor, Theresa Raimondo, has been closely working with me from the beginning and has been an invaluable source of experience and guidance. Funds The funds thus far have been used to cover my time working in the lab since the beginning of the semester, in lieu of my usual part-time semester jobs.
  4. 4. Theresa Rizk Mooney Lab for Cell and Tissue Engineering PI: Dr. David Mooney Mentor: Theresa Raimondo References: 1. Cotran; Kumar, Collins (1998). Robbins Pathologic Basis of Disease. Philadelphia: W.B Saunders Company. ISBN 0-7216-7335-X. 2. Kim, S., & Choi, I.-H. (2012). Phagocytosis and Endocytosis of Silver Nanoparticles Induce Interleukin-8 Production in Human Macrophages. Yonsei Medical Journal, 53(3), 654–657.