1. Theresa Rizk
Advisor: Theresa Raimondo
Faculty Mentor: David Mooney, Ph.D.
Nanoparticle Directed Macrophage Polarization: Exploring the Role of Phagocytosis and
Endocytosis
Background and Objective
Excessive inflammation and imbalanced macrophage activation are critical events in the
pathogenesis of many chronic inflammatory diseases such as atherosclerosis, inflammatory
bowel disease, asthma, rheumatoid arthritis, osteoarthritis, and multiple sclerosis. Acute
inflammation is a protective response that kills invading pathogens, and is further beneficial by
enhancing tissue healing provided a degree of self-limitation. However, uncontrolled activation
of recruited immune cells, particularly macrophages, leads to chronic inflammation which
counteractively impedes the proper diffusion of cells involved in the healing process, resulting in
tissue damage. Classically-activated macrophages (M1) promote inflammation through the
release of several proinflammatory signals including inflammatory cytokines, reactive oxygen
species, proteases and antimicrobial peptides. Contrastingly, alternatively-activated macrophages
(M2) serve a regulatory function as anti-inflammatory cells (M2c), and play a key role in tissue
healing (M2a). Hence, the development of therapeutics that can target inflammation and restore
proper macrophage homeostasis are of considerable interest in tissue healing and regeneration.
Current research in Dr. Mooney’s lab is focused on the design of nanoparticles capable of
directing macrophage polarization towards the M2a phenotype. Initial data suggest that IL-4
conjugated gold nanoparticles promote macrophage polarization towards the M2a phenotype in
vivo, in an animal model of ischemic inflammation, and result in improved functional recovery
of muscle strength following ischemic injury. The aim of my research this semester is to study

2. the mechanism by which the nanoparticles direct macrophage polarization. More specifically, I
will study the role of macrophage phagocytosis in nanoparticle-directed polarization. This
understanding will be used to direct the next iterations of nanoparticle design. Determining the
signaling mechanism by which phagocytosis is triggered can then be applied to increase or
decrease phagocytosis appropriately such that polarization towards the M2a phenotype is
optimized.
Detailed Plan for Research and Time frame:
Experiment outline:
(1) Develop assay to quantify gold nanoparticle (AuNP) phagocytosis (~2 weeks)
a. AuNP concentration (number of particles/mL) can be calculated using the Beer-
Lambert law. To make this calculation we will need to measure the size of the
particles with dynamic light scattering (DLS) and the absorbance of the particles
using the UV-vis spectrometer.
b. Learn how to make AuNP’s, DLS measurements and UV-vis measurements and
calculate AuNP concentration.
c. Make a standard curve of various AuNP concentrations in cell media to determine
(1) our detection limit (for how few AuNP’s we can measure) and (2) how well
we will be able to quantify the AuNP concentration. In future experiments we will
collect the media from the cell culture and calculate the AuNP concentration. We
will know how much the macrophages phagocytosed by comparing the amount of
AuNP’s in the media at the end of the culture to the amount that we initially
added to the culture.

3. (2) Use cytochalasin D, chlorpromazine, and nystatin to inhibit macropinocytosis, clathrin-
dependant endocytosis, and caveolae-dependant endocytosis respectively (~2 weeks,
macrophage culture takes about 2 weeks for each experiment)
a. Treat macrophage cell cultures with the drugs listed above, or no drug as a
control. Measure how many AuNP’s are phagocytosed in 24hrs and measure cell
viability to ensure that drug concentrations do not minimize cell viability. We will
also collect these cells and stain them for markers of M1 and M2 macrophage
polarization, to make sure that the drugs do not interfere with polarization.
(3) Use drugs listed in (2) to inhibit the various types of phagocytosis and assess macrophage
polarization (2 months)
Experimental Design
No drug cytochalasin D chlorpromazine nystatin
Nanoparticle
with IL4
Nanoparticle
with IL4
Nanoparticle with
IL4
Nanoparticle
with IL4
Soluble IL4 Soluble IL4 Soluble IL4 Soluble IL4
No IL4 No IL4 No IL4 No IL4
Here the hypothesis is that the nanoparticles will be more effective at promoting
macrophage polarization when phagocytosis is inhibited, but inhibiting phagocytosis will
have no effect on polarization driven by soluble IL4. We propose that when the AuNP is
effectively ‘eaten’ (phagocytosed) by a macrophage, it is no longer available to polarize
Method of
polarizing
macrophages
Treatment to inhibit various types of phagocytosis:

4. other cells, but if phagocytosis is inhibited then each AuNP (nanoparticle) will be able to
polarize more cells, as opposed to being consumed by just 1 cell.
Here we will look at different time points to see when phagocytosis becomes important,
and how long the macrophages maintain their phenotype.
Faculty Involvement:
I will have the opportunity to meet with Professor Mooney at least twice a month, when
my graduate student adviser and I meet with him about our project and its progress. I will
also have the opportunity to meet with him once at the beginning and once at the end of
the semester to both establish the project and its expectations, and report on the
experience and progress afterwards. Between meetings with Professor Mooney I will be
closely supervised by Theresa Raimondo, a Graduate student and Ph.D. candidate in the
lab.
Funds:
Funds will be used to cover the student’s wages for 12 hours of research per week for 12
weeks.