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Mansoor M. Amiji, PhD, RPh
Distinguished Professor and Chair
Pharmaceutical Sciences Dept.
School of Pharmacy
Co-Director, Nanomedicine
Education and Research
Consortium (NERC)
Northeastern University
Room 110 Mugar Building
Boston, MA 02115
Tel. 617-373-3137
Fax. 617-373-8886
Email: [email protected]
MEMORANDUM
TO: Drs. Jonghan Kim and Zhenfeng Duan
FROM: Mansoor M. Amiji, PhD (MA)
Distinguished Professor and Chair
RE: Adwait Oka’s MS Thesis Defense
DATE: December 5, 2014
Attached please find a copy of Adwait Oka’s MS dissertation entitled " Targeted
siRNA Delivery Strategy with Water-in-Oil-in-Water Multiple Emulsion for Modulation
of Tumor-Associated Macrophage Polarity in Immunotherapy of Cancer " for your
review.
Also, please be reminded that his MS thesis defense meeting is scheduled for
Tuesday, December 2nd, 2014 from 1 pm in conference room 230 of 140 The
Fenway Building.
Thank you.
Cc: Adwait Oka
1
Targeted siRNA Delivery Strategy with Water-in-Oil-in-Water
Multiple Emulsion for Modulation of Tumor-Associated
Macrophage Polarity in Immunotherapy of Cancer
Master’s Thesis
by
Adwait Oka
Advisor: Mansoor M. Amiji, PhD
Department of Pharmaceutical Sciences
Northeastern University
December 2nd, 2014
2
ACKNOWLEDGEMENTS
I take this opportunity to thank all those who directly or indirectly have helped me during the
course of this Thesis.
First and foremost, I would like to extend my deepest gratitude to my advisor Dr. Mansoor Amiji
for his patience, enthusiastic guidance, unending encouragement and timely critique during the
course of this research work. I am thankful to Dr. Amiji for believing in me and providing me an
opportunity to undertake this project. I am especially thankful to him for always encouraging me
especially when experiments were not working as expected and providing unrelenting support
without which this thesis would not have been possible.
I would also like to thank my committee members Dr. Jong-han Kim and Dr. Zhenfeng Duan
for their valuable time, inputs and suggestions.
I extent my special thanks to Amit, Vanessa, Meghna and Huyen for helping me with different
experimental methods and techniques. I also wish to acknowledge the timely inputs provided by
Shardool, Husain, Sunita, Lipa and Dipti in various experiments.
I am thankful to Ankita, Aatman, Purva and Srujan for guiding me and giving me company while
performing experiments.
Most importantly I will like to thank my parents who have been a constant source of motivation
and support. Their love, encouragement and sacrifices enabled me further my aspirations and
realize my dreams. I will also like to thank my relatives and friends who have supported my
decisions and encouraged me throughout my life.
Lastly, I want to thank Northeastern University, Bouvé College of Health Sciences, School of
Pharmacy and Department of Pharmaceutical for providing excellent infrastructural facilities for
conducting research.
3
TABLE OF CONTENTS
1. ABSTRACT .............................................................................................................................. 12
2. OBJECTIVES AND SPECIFIC AIMS .................................................................................... 13
2.1 Statement of the Problem .................................................................................................... 13
2.2 Objectives and Experimental Hypothesis ........................................................................... 13
2.3 Specific Aims ...................................................................................................................... 14
3. BACKGROUND AND SIGNIFICANCE ................................................................................ 15
3.1. Solid Tumor and Microenvironment .................................................................................. 15
3.2 Tumor-Associated Macrophages (TAM) ............................................................................ 18
3.3 TAM and their Role in Cancer ............................................................................................ 19
3.3.1 Inflammation-Initiated Genetic Alterations.................................................................. 20
3.3.2 Angiogenesis ................................................................................................................ 21
3.3.3 Lymphangiogenesis ...................................................................................................... 21
3.3.4 Metastasis ..................................................................................................................... 22
3.3.5 Immune Suppression .................................................................................................... 23
3.4 TAM-Targeted Therapies .................................................................................................... 24
3.4.1 Inhibition of monocyte and macrophage recruitment…………………………………25
3.4.2 Monocyte/macrophage ablation ................................................................................... 25
3.4.3 TAM re-programming/TAM repolarization. ................................................................ 26
3.4.4 Upregulation of TAM phagocytosis. ........................................................................... .28
4
3.4.5 Adoptive Transfer…………………………………………………………………….28
3.5 RNA Interference for Modulation of Macrophage Phenotype............................................ 29
3.6 Multiple Emulsions as a Macrophage-Targeted Delivery System ...................................... 32
4. EXPERIEMTNAL DESIGN AND METHODS ...................................................................... 33
4.1 Development of Macrophage Polarization Model .............................................................. 33
4.1.1 Cell Culture Conditions ................................................................................................ 33
4.1.2 Expression of Phenotype-Specific Marker Genes by RT-PCR .................................... 34
4.1.3 Expression of Phenotype Specific Marker Genes by Flow Cytometry ........................ 36
4.1.4 Preparation and Characterization of Nanoemulsions ................................................... 37
4.2 Polarization Strategies ......................................................................................................... 37
4.2.1 Polarization of J77A.1 and RAW 264.7 macrophages using LPS ............................... 39
4.2.2 Polarization of J77A.1 macrophages using Omega 3/6 Fatty Acid Nanoemulsion ..... 39
4.2.3 Polarization of J77A.1 and RAW 264.7 macrophages using EPA and RvD1 ............. 39
4.2.4 Polarization of THP-1 Monocytes using LPS+ IFN and IL-4 ...................................... 40
4.2.5 Polarization of THP-1 Monocytes using Azithromycin (AZM) .................................. 40
4.2.6 Polarization of J77A.1 macrophages using Azithromycin ........................................... 40
4.2.7 Polarization of J77A.1 macrophages using IL-4: ......................................................... 40
4.3 Formulation and Characterization of siRNA encapsulating W/O/W Multiple Emulsion ... 41
4.3.1 Formulation of Blank and siRNA encapsulating W/O/W Multiple Emulsion ............. 41
4.3.2 Size and Zeta Potential Measurement for Blank and siRNA encapsulating ME ......... 42
5
4.3.3 Qualitative Determination of siRNA Encapsulation by ME ........................................ 42
4.3.4 Quantitative Determination of siRNA Encapsulation by ME ...................................... 43
4.3.5 Determination of Stability of Encapsulated siRNA…………………………………...43
4.4 Gene Silencing and Cellular Uptake of ME encapsulated CSF-1R siRNA ........................ 44
4.5 Amplification, Purification, Isolation and Characterization of Plasmid DNA ................... 45
4.5.1 Amplification, Purification and Isolation of Plasmid DNA ......................................... 45
4.5.2 Characterization of Plasmid DNA ................................................................................ 46
4.6 Formulation and Characterization of plasmid encapsulating W/O/W Multiple Emulsion . 47
4.6.1 Formulation of Blank and Plasmid encapsulating W/O/W ME ................................... 47
4.6.2 Size and Zeta Potential Measurement for Blank, Null Plasmid and miR-155 Plasmid
encapsulating ME .................................................................................................................. 47
4.6.3 Quantitative Determination of Plasmid DNA Encapsulation: ...................................... 47
4.6.4 Stability Determination of Encapsulated Plasmid & DNAse Protection Assay ........... 48
4.7 Cellular Uptake ................................................................................................................... 48
4.8 Quantitative Determination of Intracellular miR-155 Production ...................................... 49
4.9 Gene Expression & Macrophage Re-polarization Analysis ................................................ 50
4.10 Cytotoxicity Analysis ........................................................................................................ 51
5. RESULTS AND DISCUSSION ............................................................................................... 52
5.1 Macrophage Polarization Strategies .................................................................................... 52
5.1.1 Polarization of J77A.1 and RAW 264.7 macrophages using LPS ............................... 52
6
5.1.2 Polarization of J77A.1 macrophages using Omega 3/6 Fatty Acid Nanoemulsion ..... 52
5.1.3 Polarization of J77A.1 and RAW 264.7 macrophages using EPA and RvD1 ............. 54
5.1.4 Polarization of THP-1 Monocytes using LPS+ IFN and IL-4 ...................................... 54
5.1.5 Polarization of THP-1 Monocytes using Azithromycin (AZM) .................................. 56
5.1.6 Polarization of J77A.1 macrophages using Azithromycin ........................................... 56
5.1.7 Polarization of J77A.1 macrophages using IL-4: ......................................................... 57
5.2 Characterization of Blank and siRNA encapsulating W/O/W Multiple Emulsion ............. 58
5.2.1 Size and Zeta Potential Measurement for Blank and siRNA encapsulating ME ........ 58
5.2.2 Qualitative Determination of Encapsulation by ME .................................................... 58
5.2.3 Quantitative Determination of siRNA Encapsulation by ME ...................................... 59
5.2.4 Determination of Stability of Encapsulated siRNA ..................................................... 59
5.3 Gene Silencing and Cellular Uptake of ME encapsulated CSF-1R siRNA ........................ 60
5.4 Characterization of Plasmid DNA....................................................................................... 63
5.5 Characterization of Plasmid encapsulated W/O/W Multiple Emulsion .............................. 64
5.5.1 Size and Zeta Potential Measurement for Blank, Null Plasmid and miR-155 Plasmid
encapsulating ME .................................................................................................................. 64
5.5.2 Quantitative Determination of Plasmid DNA Encapsulation ....................................... 65
5.5.3 Stability Determination of Encapsulated Plasmid & DNAse Protection Assay ........... 66
5.6 Cellular Uptake ................................................................................................................... 66
5.7 Quantitative Determination of Intracellular miR-155 Production ...................................... 68
7
5.8 Gene Expression & Macrophage Re-polarization Analysis ............................................... 68
5.9 Cytotoxicity Analysis.......................................................................................................... 72
6. CONCLUSIONS....................................................................................................................... 73
7. REFERENCES ......................................................................................................................... 74
8
LIST OF FIGURES
Figure 1: TME and its cellular components .................................................................................. 16
Figure 2: Stimulants, specific markers and functional characteristics of different macrophage
phenotypes .................................................................................................................................... 19
Figure 3: TAMs and their role in cancer pathogenesis ................................................................. 20
Figure 4: TAM involvement in Angiogenesis .............................................................................. 22
Figure 5: Microenvironmental components in the process of Metastasis..................................... 23
Figure 6: TAM initiated immunosuppression. .............................................................................. 24
Figure 7: TAM-targeted Therapeutic Strategies ........................................................................... 25
Figure 8: Biogenesis and Mechanism of siRNA and miRNA induced interference. ................... 30
Figure 9: Diagrammatic representation of W/O/W Multiple Emulsion. ...................................... 32
Figure 10: Diagrammatic representation of process of preparation of w/o/w ME ....................... 41
Figure 11: Methodology for Plasmid Amplification, Purification and Isolation .......................... 46
Figure 12: Experimental Scheme for Cytotoxicity Study ............................................................. 51
Figure 13: Semi-quantitative analysis of M1 and M2 specific gene expression after 6 hour LPS
stimulation (100 ng/ml)................................................................................................................. 52
Figure 14: Semi-quantitative analysis of M1 and M2 specific gene expression after 6 and 12 hour
treatment with different nanoemulsions........................................................................................ 53
Figure 15: Semi-quantitative analysis of M1 and M2 specific gene expression after 24 and 48
hour treatment with different nanoemulsions ............................................................................... 53
Figure 16: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression after
treatment with EPA and RvD1 ..................................................................................................... 54
9
Figure 17: Semi-quantitative analysis of M1 phenotype specific gene expression after M1 and
M2 polarizing stimuli in THP-1 cells ........................................................................................... 55
Figure 18: Semi-quantitative analysis of M2 phenotype specific gene expression after M1 and
M2 polarizing stimuli in THP-1 cells ........................................................................................... 55
Figure 19: Expression of Surface markers of M1 and M2 phenotypes after M1 and M2 polarizing
stimuli in THP-1 cells ................................................................................................................... 55
Figure 20: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression after
LPS and IFN treatment in the presence and absence of AZM in THP-1 cells ............................. 56
Figure 21: Expression of Surface markers of M1 and M2 phenotypes after AZM treatment in
J774A.1 cells ................................................................................................................................. 56
Figure 22: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression after
LPS and IFN treatment in the presence and absence of AZM in J774A.1 cells ........................... 57
Figure 23: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression after
16h IL-4 (100ng/ml) Stimulation in J774A.1 cells ....................................................................... 57
Figure 24: Bright-field image showing oil globules having diameter of 1.0 um or less and
Fluorescent Microscopy image of a stable non-leaky ME encapsulating dextran conjugated
tetramethyl-rhodamine .................................................................................................................. 58
Figure 25: Standard Curve for PicoGreen Assay for siRNA Encapsulation ................................ 59
Figure 26: Agarose Gel Image showing absence of siRNA in external aqueous phase of W/O/W/
multiple emulsion.......................................................................................................................... 59
Figure 27: Agarose Gel Image showing the stability of extracted siRNA ................................... 60
Figure 28: Quantitative Analysis of CSF-1R expression 24 & 48 hour after treatment with
different concentrations of siRNA ................................................................................................ 60
10
Figure 29: Quantitative CSF-1R Expression Analysis 24 and 48 hours post various treatments. 61
Figure 30: Cellular Uptake of Alexa 647 labelled siRNA encapsulated in multiple emulsion post
1, 2 and 4 hours of incubation in J774A.1 macrophages .............................................................. 62
Figure 31: Figure 31: (A & B) Null and miR-155 encoding expression plasmid size and features
(C) miR-155 encoding insert with restriction site sequence for BamHI and NheI ....................... 63
Figure 32: Agarose Gel Image showing the size of Null and miR-155 Plasmid and miR-155
insert after restriction Digestion.................................................................................................... 64
Figure 33: Standard Curve for PicoGreen Assay for Plasmid Encapsulation .............................. 65
Figure 34: Agarose Gel Image showing stability and protection against DNAse treatment of
encapsulated Plasmid .................................................................................................................... 66
Figure 35: Fluorescence and Brightfield Images showing uptake of multiple emulsion
encapsulating plasmid 1, 2, 4 and 6 hour post incubation. ........................................................... 67
Figure 36: Quantitative Analysis of Intracellular miR-155 levels under various conditions ....... 68
Figure 37: Quantitative Determination of IL-1β expression after various treatments 12, 24 and 48
hours post IL-4 Stimulation .......................................................................................................... 69
Figure 38: Quantitative Determination of iNOS expression after various treatments 12, 24 and 48
hours post IL-4 Stimulation .......................................................................................................... 70
Figure 39: Quantitative Determination of Arg-1 expression after various treatments 12, 24 and 48
hours post IL-4 Stimulation .......................................................................................................... 71
Figure 40: Cytotoxicity Analysis of ME System in J77A.1 macrophages 24 and 48h after
treatment ....................................................................................................................................... 72
11
LIST OF TABLES
Table 1: Immune Cells in TME and their functional roles. .......................................................... 17
Table 2: Clinically approved or under trial drugs targeting TAMs .............................................. 29
Table 3: Polarization approaches investigated in different cell lines ........................................... 38
Table 4: Size and Zeta Potential Measurements of Blank and siRNA encapsulated ME ............. 58
Table 5: Size and Zeta Potential for Blank, Null Plasmid and miR-155 Plasmid encapsulating
ME................................................................................................................................................. 64
Table 6: Encapsulation Efficiency of mir-155 and Null Plasmid ME .......................................... 65
.
12
1. ABSTRACT
Despite greater understanding of the molecular mechanisms and the resulting targeted
therapeutic strategies, the mortality rate associated with cancer has not changed to a great extent.
Clinical and experimental data suggests that the components of the tumor microenvironment
(TME) especially Tumor-associated macrophages (TAMs) are the real players driving cancer
progression via functional support to cancer cells by playing a central role in every stage of cancer
development –initiation, growth and proliferation, angiogenesis and finally metastasis. Thus,
utilizing the re-programming/ re-education approach targeting TAM’s can serve as a promising
therapeutic target in the fight against the cancer.
The preliminary studies in J77A.1 showed that macrophages can be polarized to M1
phenotypic state after LPS stimulation and M2 phenotypic after IL-4 stimulation. Water in oil in
water (w/o/w) multiple emulsions (ME) encapsulating siRNA were formulated and the oil droplet
size, zeta potential and encapsulation efficiency was found to be 0.8 µ, -46 to -47mV and 99.7%
respectively. Qualitative determination of siRNA encapsulation confirmed stability of siRNA after
encapsulation. Though the siRNA was taken up by cells it could not silence expression of CSF-1R
and so use of microRNA based RNA interference was chosen as a possible approach.
ME formulated using miR-155 encoding plasmids protected the plasmid against DNAse
activity. The plasmids after encapsulation maintained its integrity and ME had an oil droplet size
of 0.9µ, encapsulation efficiency of 60-65% and zeta potential of -46 to -47mV. A time dependent
uptake of emulsions was seen and a 3 fold increase in intracellular expression of miR-155 was
achieved which translated into upregulation of M1 marker genes (IL-1β and iNOS) indicating
polarization to M1 phenotype. The emulsions were not cytotoxic and were well tolerated by the
macrophages.
13
2. OBJECTIVES AND SPECIFIC AIMS
2.1 Statement of the Problem
Even after great strides have been made in cancer research, development of a safe and
successful clinical therapy has still not been achieved. The major hurdle in cancer management
using traditional methods of radiation and chemotherapy as well as the newer targeted therapies is
the relapse and refractoriness due to the metastatic potential [1]. Metastasis also is the major reason
for high mortality associated with cancer [2]. Thus it is extremely essential to develop a treatment
regimen that can target the components nurturing this metastatic potential. It has been established
that cancer cells and the microenvironmental components influence each other’s functions and are
responsible for the cancer pathogenesis [3]. Stromal microenvironment is prominently populated
by macrophages called as tumor-associated macrophages (TAM) in almost all types of malignant
conditions [4]. These TAMs recruited and residing in the tumor microenvironment are the major
players that are involved in the almost every stage of cancer development starting from initiation
to growth, progression and metastasis. On account of their central role in cancer etiology, TAMs
have attracted a lot of attention as a target for anti-tumor therapy [5]. Out of the various strategies
available for targeting TAM for anticancer action, one strategy of macrophage re-education/ re-
polarization/ re-programming is extremely promising. This re-programming or phenotype
switching strategy aims at targeting the TAMs that express the M2 polarized state and would spare
the tumoricidal M1 polarized macrophages. This strategy will be used to selectively re-polarize
the M2 differentiated TAMs back to anti-tumor M1 phenotypic macrophages.
2.2 Objectives and Experimental Hypothesis
The main objective of this project was develop a macrophage targeted delivery system for
encapsulation, stabilization and delivery of siRNA to ascertain if the macrophage phenotype can
14
be switched from M2 (pro-tumor) to M1 (anti-tumor) polarized state. In order to achieve this, the
first objective was to evaluate the expression of macrophage phenotype specific marker genes in
the presence of various polarizing stimuli using RT-PCR and FACS so as to develop a stable and
reproducible model for investigating the phenotype modulation. The second objective was to
formulate CSF-1R and IL-10 siRNA encapsulating w/o/w multiple emulsion (ME) system having
oil droplet size of around 1 micron that would selectively be taken up by J774A.1 macrophages.
The hypothesis was that TAM polarization to M2 phenotype is essential for tumor growth
and metastasis. The re-polarization of these TAMs to M1 phenotype using a targeted delivery
system for promotion of RNA interference can be a strategy to achieve anti-tumor activity.
2.3 Specific Aims
Aim 1: Development of Macrophage Phenotype Modulation Model
a) Assessment of macrophage phenotype specific marker gene expression using RT-PCR and
FACS under baseline conditions
b) Evaluation of change in macrophage phenotype specific marker gene expression in response
to lipopolysaccharide (LPS) for M1 and azithromycin (AZM) for M2 phenotype
Aim 2: Formulation and Characterization of Blank and siRNA Encapsulated Water-in-Oil-
in Water (W/O/W) Multiple Emulsion
a) Preparation of Blank and siRNA encapsulated W/O/W ME using safflower oil
b) Characterization of blank and siRNA encapsulated W/O/W ME with respect to size, zeta
potential.
c) Determination of encapsulation efficiency and stability of encapsulated siRNA.
Aim 3: Assessment of Macrophage Phenotype Switching from M2 to M1 upon siRNA
Administration in Control and W/O/W Multiple Emulsion Formulations
15
a) Evaluation of Cellular Uptake and Trafficking of encapsulated siRNA
b) Determination of gene knockdown and change in macrophage phenotype specific marker
gene expression.
c) Investigation of cytotoxicity of siRNA and formulation
3. BACKGROUND AND SIGNIFICANCE
Cancer is one of the foremost public health concern not only in the United States, but also
around the world [6]. Cancer still remains one of the most deadly diseases in the world and has
been estimated to account for 13.1 million deaths out of the 22 million diagnosed cases each year
by 2030 [7]. In the United States, cancer is ranked as the second most common cause of mortality
accounting for 1 out of every four deaths. [6]. The American Cancer Society had predicted
diagnosis of about 1,665,540 new cancer cases in the United States in 2014 of which deaths in
about 585,720 cases have been predicted at a rate of 1,600 deaths per day [8]. Thus to ensure safe
and effective management of cancer, finding new targets and treatment regimens is the need of the
hour.
3.1. Solid Tumor and Microenvironment
The National Cancer Institute defines solid tumors as “an abnormal mass of tissue that
usually does not contain cysts or liquid areas”. It had been assumed that solid tumors are a
collection of homogeneous aneuploid cells that have a potential to migrate to other organs.
However it has been realized that these are organ like assemblies which are structurally complex
consisting of an eclectic mixture of multiple cellular entities belonging to different cell lineages
16
that together form the tumor microenvironment (TME) [9, 10].
Figure 1: TME and its cellular components. Reproduced from reference [11]
The fact that tumor cells are solely responsible for the growth and progression of tumors
has been dispelled by recent experimental and clinical data which reveals that, for the pathogenesis
of cancer, the cancer cells are totally dependent on the functional support provided by the adjacent
non-tumoral cells [3]. The tumor microenvironment is an intricate and dynamic milieu of
numerous cellular components like endothelial cells, smooth-muscle cells, fibroblasts of various
phenotypes, myofibroblasts, mast cells, pericytes and immune cells like T, B and natural killer
lymphocytes, antigen presenting cells (APC) such as macrophages and dendritic cells, neutrophils
and other granulocytes (eosinophils and basophils) [12]. The interplay of these component with
one other, with the extracellular matrix (ECM) components and along with the cues provided by
signaling molecules and soluble factors in the vicinity drives the cancerous transformation,
invasion, growth and metastasis while inhibiting host immune response and providing therapeutic
resistance [13]. Every component of the microenvironment has a specific role to plays in
tumorigenesis.
17
Table 1: Immune Cells in TME and their functional roles. Reproduced from [14]
Abbreviations: TP - Tumor Progression; TR- Tumor Regression
Cell Type Infiltrating
Population Outcome Function
Macrophages
M1
M2
TR
TP
Activation of immune responses by MyDD88/TLRpathways
Promotion of angiogenesis; suppression of CTL function; Recruitment of
CCR6+ TREG; positive modulation of the tumorigenic and angiogenic
potential of CSC
T
lymphocytes
CTL
Treg
Γδ T cells
TR
TP/TR
TP/TR
Specific tumor cell killing activity
TP: functional suppression of CTL, DC, NK cells and macrophages.
TR: correlation with good prognosis in some solid tumors, hypothetically due
to lacking of suppressor activity and other unidentified activities
TP: inhibition of CTL and NK cell activity; promotion of angiogenesis
TR: cytotoxic activity, IFN-γ production
Dendritic
cells
CD8a+ DC
Plasmacytoid
DC
TIM-3C DC
TR
TR
TP
Processing and presentation of soluble tumor-associated antigens; Type I IFN-
dependent CD8+ T cell cross-priming against antigens released from dying
tumor cells
Processing and presentation of soluble tumor associated antigens
Suppression of HMGB1-dependent innate immune responses
NK cells
NKp46+
NKp30+
DNAM-1+
CD69+
CD155+
TR Specific tumor cell killing activity by secretion of perforin and granzyme B-
containing granules as well as release of calcium ions; DC editing; killing
activity against CSC
NKT cells
NK receptors
TCR-a chain
variants
CD1d-
restricted
CD57+
TP/TR TP: CD1d-restricted cytotoxic activity; IFN-γ production; APC stimulation
TR: Th2 cytokine production
Myeloid-
derived
Suppressor
cells
CD11b+ Gr-
1+
TP Repression of the effect or function of T lymphocytes and NK cells; highly
present in late stages of tumor progression; promotion of TREG functions;
promotion and sustainment of angiogenesis; present in elevated number in
highly aggressive microenvironments
Cancer stem
cells
CD34+
CD133+
TP Self-renewal function; tumor initiating activity; promotion and sustainment of
angiogenesis; tumor resistance; Sustainment of the tumor mass
18
Immune cells are functional in almost every step of carcinogenesis promoting cell
proliferation, tissue remodeling, angiogenesis and metastasis [9]. Cancer associated fibroblasts
(CAF) promote synthesis, disposition and ECM remodeling and also support vessel development
via production of CXCL12 which promotes recruitment of marrow derived precursors [9, 15].
Endothelial cells are involved in the formation of new blood vessels from existing vessels.
Pericytes play a role in stabilizing and maturation of newly formed vessels by promoting
endothelial cell survival [16]. Adipocytes are responsible for secretion of various factors that
intensify the inflammatory and angiogenic process [17]. ECM components control the tumor
stiffness via tissue remodeling and promote the motility and migration potential of the cancerous
mass [16]. Thus cancer cells along with the microenvironmental components need to be considered
as a combined dysfunctional entity and treatment approaches need to be directed at both the cancer
cells as well as components in the tumor microenvironment.
3.2 Tumor-Associated Macrophages (TAM)
Leukocytic infiltration is a distinguishing characteristic of a diverse set of pathological
conditions and also is a hallmark of tumors [18]. Macrophages form the major subset of this
leukocytic infiltrate and the number of macrophages can be up to 65%, though this varies from
tumor to tumor [19] These macrophages in the leukocytic infiltrate can be skewed to different
phenotypic states depending on the environmental cues provided and can exhibit a pleotropic
nature of both tumor-promoting and tumor-inhibitory actions [19] . The macrophages can exhibit
extremes of a functional continuum namely an anti-tumor/ M1 phenotype or pro-tumor/ M2
phenotype. The various phenotypes and the characteristics of these have been demonstrated in the
Figure 2 below:
19
Figure 2: Stimulants, specific markers and functional characteristics of different
macrophage phenotypes. Reproduced from reference [20]
The M1 macrophages are the ones that are involved in destruction of micro-organisms and
tumor cells by producing pro-inflammatory cytokines. The M2 macrophages are involved in
scavenging debris, tissue remodeling and repair and angiogenesis. Based on these functional roles,
it can be clearly seen that the macrophages in the microenvironment are polarized towards the pro-
tumor/ M2 phenotype and this subpopulation is commonly referred to as tumor-associated
macrophages (TAM) [21]. TAMs are extremely crucial for the pathogenesis of cancer since these
are involved in all stages of cancer namely growth, angiogenesis, progression and proliferation,
and metastasis.
3.3 TAM and their Role in Cancer
TAMs are one of the major components of the tumor microenvironment which are as
important as the tumor cells in the pathogenesis of cancer. TAMs orchestrate every stage of cancer
20
starting from immunosuppression to angiogenesis to growth and proliferation and finally
metastasis [22].
Figure 3: TAMs and their role in cancer pathogenesis. Reproduced from reference [23]
Experimental and clinical data suggests that TAMs play a role in enhancing cancer’s radio-
protective effects and drug resistance and are also responsible for treatment failure and poor
prognosis in cancer patients [24] In the initial stages of tumorigenesis, TAMs produce high
amounts of inflammatory factors that play a role in neoplastic transformation whereas after tumor
establishment, the secrete mediators that contribute to immunosuppression, angiogenesis, tissue
remodeling, progression and metastasis. The mechanisms involved in all these processes are as
follows,
3.3.1 Inflammation-Initiated Genetic Alterations
It has been elucidated that chronic and untreated inflammation is the underlying cause in
many cancers [5, 25]. In response to infections or tissue insult, macrophages get recruited and
21
release a number cytokines and chemokines which intensify the inflammatory process [26]. The
reactive nitrogen and oxygen moieties produced during this process can damage DNA causing
genetic alterations and instability. The accumulation and stabilization of these mutations in the
absence of DNA repair leads to initiation of cancer [27, 28]. Once initiated, the tumor cells secrete
tumor derived factors like M-CSF and CCL2 which promote recruitment of more macrophages
and monocytes [22].
3.3.2 Angiogenesis
Clinical evidence suggests that there is positive correlation between TAM density and
angiogenesis [29]. Tumor development begins with an avascular phase which switches to a
vascular phase as the tumor grows. This transition called as the “angiogenic switch” is
orchestrated by TAMs which release a variety of soluble mediators like fibroblast growth factor
(FGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), platelet
derived growth factor (PDGF), thymidine phosphorylase (TP), angiopoietins (ANG1 and ANG2),
transforming growth factor (TGF-β), TNF-α, IL-1β, IL-8, nitric oxide (NO) [22, 29, 30] They also
release a number of proteolytic enzymes which are responsible for ECM and tissue remodeling
like matrix metalloproteinases (MMP-1, 2, 3, 7, 9, 12), cathepsin B, urokinase plasminogen
activator (uPA), matrix remodeling enzymes like lysyl oxidase, SPARC. The temporal and spatial
expression of these molecules promotes proliferation and migration of endothelial cells,
remodeling of ECM and ultimately formation of stable vessels. [31].
3.3.3 Lymphangiogenesis
Formation of leaky lymph vessels is another mode by which tumor cells metastasize. It has
been hypothesized that macrophages concomitantly promote the processes of lymphangiogenesis
and angiogensesis. Both tumor cells and TAMs induce lymphangiogenesis by expression of
22
lymphangiogenic factors like VEGF-C and VEGF-D which activate the VEGF receptor 3 on
growing lymph vessels and thus promote formation of lymphatic vascular network [29]. Recent
evidence has proposed that TAMs not only promote lymphatic vessel formation through secretion
of paracrine mediators but also by direct involvement in vessel formation by differentiating into
lymphatic endothelial cell progenitors (LECP) which provide structural and directional support to
vessel formation [32]
Figure 4: TAM involvement in Angiogenesis. Reproduced from reference [33]
3.3.4 Metastasis
Clinical studies have established a relationship between TAMs and tumor metastasis.
TAMs are responsible for all steps of metastasis starting from migration and invasion of tumor
cells to intravasation to extravasation and establishment of metastasis [22]. Migration and invasion
of tumor cells and macrophages occurs simultaneously which causes them to come in contact with
blood and lymph vessels. Tumor cells secrete M-CSF which stimulates macrophage migration
whereas macrophages release EGF which promotes tumor cell migration [34]. Macrophages
23
through the action of protease degrade the ECM allowing tumor cell escape via the process of
invasion [5]. TAMs via secretion of TNF-α increase the intravasation i.e. entry of tumor cells into
blood. This occurs via clusters of macrophage cells at the boundary between tumor and vessels
[35, 36]. TAMs secrete VEGF which causes local vascular permeability that assists in tumor cell
extravasation. Also binding of TAM expressed α4β1 integrin with tumor cell expressed vascular
cell adhesion molecule-1 (VCAM-1 or CD106) leads to tethering of tumor cells to macrophages
and thus prevents apoptosis [22, 31].
Figure 5: Microenvironmental components in the process of Metastasis. Reproduced from
reference [11]
3.3.5 Immune Suppression
Experimental data has revealed that macrophages also play a crucial role in suppressing
the host immune response. The tumor cells release certain TDF’s which cause TAMs to lose their
ability of antigen presentation and stimulation of T and natural killer (NK) cells [21]. This effect
is via enhancing the expression of enzymes, cytokines and chemokines like arginase, indoleamine
deoxigenase (IDO) metabolites, prostaglandins, IL-10 and TGF-β, CCL18 that stimulate inactivity
24
of antigen presenting cells, B and T cells [21, 29]. Arginase causes decrease in levels of L-arginine
in TME thereby changing the alteration in expression of T cell receptors and its signaling capacity.
TAMs by secreting CCL22 trigger activation of regulatory T cells which are involved in
immunosuppression via blocking the activity of effector T and other inflammatory cells [29]. A
diagrammatic representation of TAM role in immunosuppression is shown in Figure 6 below:
Figure 6: TAM initiated immunosuppression. Reproduced from reference [21]
3.4 TAM-Targeted Therapies
Thus it is clear that TAMs are an integral component of the tumor microenvironment and
play a crucial role in every stage of cancer pathogenesis right from invasion to progression to
metastasis. Thus targeting these pro-tumoral components can prove to be important strategy in
cancer management and therapy. Anti-tumor TAM targeted strategies can be divided into 5 distinct
methodologies: (1) inhibition of monocyte and macrophage recruitment (2) monocyte/macrophage
ablation (3) TAM re-programming/ TAM repolarization (4) Upregulation of TAM phagocytosis
and (5) Adoptive transfer [24] [33] [22].
25
Figure 7: TAM-targeted Therapeutic Strategies. Adapted from reference [24]
These strategies have been briefly described in the below,
3.4.1 Inhibition of monocyte and macrophage recruitment: The cytokines and chemokines
released by tumor and stromal cells promote the recruitment of macrophages and monocytes to the
26
site of tumor. Thus depleting these chemoattractants can prove to be beneficial for cancer therapy.
The various chemoattractants that have been implicated in monocyte/macrophage recruitment
include CCL2, M-CSF, HIF, VEGF, CXCL-12, and CCR5. Various strategies have been already
tried to target these molecules and have been shown to be efficacious in decreasing the macrophage
infiltration. Some of the examples include use of small molecules trabectidin, bindarit, siRNA and
antibodies against CCL/CCR2 [37-41] , use of antisense-RNA, small molecule inhibitors, siRNA
and monoclonal antibodies against M-CSF and its receptor M-CSFR [5,42].
3.4.2 Monocyte/macrophage ablation: Another TAM targeted strategy is to promote TAM
depletion via directly triggering apoptosis using small molecules, bacteria, antibodies targeting
TAM markers, or via induction of T-cell mediated recognition and ablation [24]. Bisphosphonates
mainly clodronate and zoledronic acid have been studied extensively to ascertain their anti-TAM
action [24]. Zoledronic acid is in fact in clinical use for treatment of breast cancer [43, 44].
Immunotoxin conjugated antibodies targeting scavenger receptor A, CD52 and folate receptor β
have also been studied for their anti-TAM effect [45-47]. Folate receptor β targeted antibodies is
a better approach since it selectively eradicates TAM/M2 macrophages which overexpress this
receptor thus leaving behind the anti-tumoral M1 macrophages [47]. Injections of certain bacterial
strains like Shigella flexneri have shown 74% tumor regression in mice model. Induction of T-cell
mediated cytotoxicity has also shown to induce TAM ablation [48]. Legumain-based DNA
vaccines which stimulate CD8+ T cell response and Cd1 overexpression using retinoic acid to
stimulate natural killer cell mediated TAM depletion have also provided promising results [49,
50].
3.4.3 TAM re-programming/TAM repolarization: It is a known fact that macrophages are
plastic cells that can be polarized to different activated states based on the environmental cues
27
available. Thus induction of the anti-tumor M1 phenotype can be another TAM based strategy for
cancer therapy. NF-κB and STAT 1 are one a couple of transcriptional regulators that promote M1
phenotype. Thus upregulation and activation of NF-κB via Toll like receptor (TLR) agonists like
PolyI:C (TLR-3), LPS and monophosphoryl lipid A (TLR 4), imiquinod and R-848 (TLR 7) and
CpG-oligodeoxymucleotide (TLR-9) and anti-CD40 and anti-IL-10R antibodies can be successful
in reprogramming or re-education of macrophages to the pro-tumor state [24]. Use of interferons
(IFN) and IFN-mimics also can be used in stimulating the M1 phenotype by activating the STAT-
1. However, great caution should be exercised while using these strategies since activation of these
transcriptional factors has also been linked to pro-tumoral activity in certain tumors [24]. Other
strategies which are under clinical trials include use of GM-CSF and IL-12 cytokines either
directly or via a gene therapy based approach and use of molecules like thymosin-α1 [22, 24].
Also, since macrophages are recruited and then stimulated to express the M2 phenotype
by the tumor cells, inhibition of these M2 polarizing signals can also be a viable alternative for
TAM reprogramming. STAT 3 and STAT 6 signaling is the major pathway promoting M2
polarized state. Inhibition of STAT 3 using small molecule inhibitors like WP1066, corosolic
acid, oleanoic acid and tyrosine kinase inhibitors like sunitinib and sorafenib have shown
reversal of immune tolerance and immune suppression [51-55]. Inhibition of upstream and
downstream mediators in STAT 6 signaling can also prevent polarization to M2 phenotype.
These targets include inhibition of phosphatidylinositol 3-kinase (PI3K), c-Myc and Kruppel-like
factor 4 (KLF4) and stabilization of Src homology 2-containing inositol-5´-phosphatase (SHIP).
Other signaling pathways and proteins whose inhibition could be crucial in macrophage
reprogramming include hypoxia induced factors (HIF’s), Ets family members (Ets2), peroxisome
proliferator-activated receptor. There are also some drugs that can be skew the phenotype to M1
28
like Histidine rich glycoprotein (HRG), Proton pump inhibitor pantoprazole (PPZ), silibinin,
cisplatin, copper chelate (CuNG), 5,6-dimethylxanthenone-4-acetic acid (MDXAA) [24].
3.4.4 Upregulation of TAM phagocytosis: CD 47 is an anti-phagocytic cell surface protein
expressed by normal cells which protects them against phagocytosis by binding to SIRPα receptor
on macrophages and dendritic cells. The tumor cells adopt a similar method to escape phagocytosis
[56]. Thus blocking this interaction between CD-47 and SIRPα can be a practical alternative to
attain anti-tumor action [57]. Antibody targeted to CD-47 protein has shown to be useful in
regression of variety of tumors [58, 59]. Use of fusion protein to inhibit SIRPα signaling can be
another strategy to achieve anti-tumor effect via upregulation of macrophage phagocytosis [60].
3.4.5 Adoptive transfer: Apart from all the strategies discussed above, the method of injecting
monocytes and macrophages, manipulated in vitro or those that can be modified in-vivo, can also
be another strategy for cancer therapy [33]. In this method, the macrophages can be activated to
the classical or M1 phenotype, by sensitizing it to LPS or IFN-γ in vitro or by sensitizing to GM-
CSF in vivo. These modified macrophages can then be injected intravenous (i.v.) or intraperitoneal
(i.p.) administration to treat tumors or metastases in the peritoneal cavity [61, 62]. Another
approach involves using macrophages as vectors to expresses genes for inflammatory cytokines or
genes to convert prodrugs into active chemotherapeutic agents within the tumor [63].
Some of the drugs targeting TAMs that have been clinically approved or under trials are
discussed in the Table 2 below:
29
Table 2: Clinically approved or under trial drugs targeting TAMs. Reproduced from
reference [24]
3.5 RNA Interference for Modulation of Macrophage Phenotype
Among the various TAM targeted strategies, macrophage re-education or reprogramming
seems to be a promising concept. This goal of repolarization can be achieved by using RNA
Interference which involves silencing the genes that promote the macrophage polarization to M2
phenotype or activating genes that promote the polarization to the M1 phenotype using either
30
siRNA or miRNA mediated strategies. The diagrammatic representation of the siRNA and miRNA
interference approach is as shown below:
Figure 8: Biogenesis and Mechanism of siRNA and miRNA induced interference.
Reproduced from references [64-66]
31
Not many investigations have looked at small interfering RNA (siRNA) mediated
modulation of macrophage phenotypes, however those that have been tried have shown promising
results. One such study investigated the effect of CSF-1R siRNA on the TAMs in a mouse model
of pro-neuronal glioblastoma multiforme (GBM). The results indicated that the silencing of CSF-
1 R did not decrease the number of TAMs but on the contrary repolarized them to M1 phenotype
as revealed by decrease in expression of M2 marker genes [67]. Another study examined the effects
of silencing of SOCS-1 on TAMs tumor promoting ability. The study revealed that silencing led
to tumor suppression via IFN-γ dependent Th1 and CTL response. This indicates that there is
probability that SOCS-1 silencing promotes the reprogramming of TAMs to the inflammatory/
anti-tumor M1 phenotype [68]. In yet another study a combination of TLR9 siRNA and CpG DNA
caused a decrease in levels of certain inflammatory cytokines. This though not investigated could
be attributed to induction of change in macrophage phenotype [69].
MicroRNA-155 (miR-155) is truly a multifunctional microRNA having crucial functions
in hematopoiesis, inflammation, cancer and immunity [70]. It has emerged as a key player in
cancer and cancer immunity development having both pro and anti-tumoral effects depending on
the cell type in which its expression is altered. It has been considered both an oncomiR and an
immunomiR which clearly suggests its pro and anti-tumoral activity respectively [71]. miR-155
upregulation has been associated with B-cell cancer and breast carcinomas [71, 72]. However miR-
155 knockdown in myeloid compartment of a breast cancer mouse model has shown to hasten
breast tumor growth. Additionally, the knockdown also resulted in the transitioning of TAM’s to
a M2/Th2 response [72].
32
3.6 Multiple Emulsions as a Macrophage-Targeted Delivery System
The multi-compartmental water in oil in water (w/o/w) multiple emulsions (ME) are
triphasic systems which consist of an internal aqueous phase which is encapsulated in oil droplets
which is further dispersed in an external aqueous phase. A schematic representation of w/o/w ME
is as shown in the figure below,
Figure 9: Diagrammatic representation of W/O/W Multiple Emulsion. Reproduced from
reference [73]
This system serves as an efficient delivery system to immune cells. The size and
dimensions of the oil droplets mimic that of most natural pathogens which improve the uptake and
phagocytosis by antigen presenting cells (APC) like macrophages [74]. This will ensure efficient
and complete release of the payload encapsulated in the internal phase. The internal aqueous phase
can serve to sustain the release of encapsulated cargo [75]. Additionally, multiple emulsions
provide the possibility of entrapping multiple payloads into different phases of the multiple
emulsion thereby improving the stability of the encapsulated agents. Additionally the use of
safflower oil in the formulation of the multiple emulsion provides added advantage of having a
slight stimulatory effect on inflammatory response. Safflower oil used contains high linoleic acid
(70%) content which is an omega 6 fatty acid that known to be a precursor for synthesis of
arachidonic acid which is involved in the synthesis of pro-inflammatory eicosanoids and other
autacoids [76].
33
4. EXPERIEMTNAL DESIGN AND METHODS
4.1 Development of Macrophage Polarization Model
Plasticity is one of the distinguishing characteristics of macrophages which enables them
to differentiate into different polarized states and phenotypes based on the environmental stimuli.
In order to ascertain if classical and alternative macrophage polarization can be achieved and to
develop a reproducible polarization model, different macrophage and monocytic cell lines such as
J774A.1, RAW 264.7 and THP-1 cell lines were subjected to different polarizing stimuli, which
included bacterial membrane components, cytokines, o/w nanoemulsions containing oils rich in
omega-3 and omega-6 fatty acid and antibiotics. RT-PCR and FACS was used to determine the
expression of phenotype specific marker genes.
4.1.1 Cell Culture Conditions
J77A.1 Murine Macrophage Cell Line: The adherent J774A.1 murine macrophage cell
line (ATCC® TIB-67™) was obtained from the American Type Culture Collection (ATCC;
Manassas, VA) and grown using Dulbecco’s Modified Eagle Medium (DMEM; Mediatech Inc.,
Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Fisher Scientific, Pittsburg, PA)
and 1% Penicillin-Streptomycin-Amphotericin B antibiotic combination (Pen/Strep/Amphotericin
B; Lonza Walkersville Inc., Walkersville, MD) in a T-25 flask at 37ºC and 5% CO2. The cells were
plated at a seeding density of 1 million cells per 5ml and allowed to grow overnight before the
polarization stimulus was added the following day.
RAW264.7 Murine Macrophage Cell Line: RAW264.7 adherent murine macrophage cell
line (ATCC® TIB-71™) was obtained from ATCC and cultured in a T-25 flask at 37ºC and 5%
CO2 using DMEM modified with 10%FBS and 1% Pen/Strep/Amphotericin B. One million cells
were plated and grown overnight with the polarization started the next day.
34
THP-1 Human Monocytic Cell Line: The THP-1 suspension monocyte cell line (ATCC®
TIB-202™) was purchased from ATCC (Manassas, VA) and cultured at density of 2x105 cells/ml
in RPMI 1640 (Mediatech Inc., Manassas, VA)media modified with 0.05mM 2-mercaptoethanol,
1% Pen/Strep/Amphotericin B and 10% FBS in a T-25 flask at 37ºC and 5% CO2. The monocytic
differentiation to macrophages was promoted by adding phorbol 12-myristate 13-acetate (PMA)
purchased from Sigma-Aldrich (St. Louis, MO) at a concentration of 5ng/ml for 2 days. Following
the differentiation the polarization studies were conducted.
4.1.2 Expression of Phenotype-Specific Marker Genes by RT-PCR
RNA Extraction: The cells were harvested and lyzed using the lysis buffer supplied with
the High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany). The total RNA
from the cell lysate was then isolated by following the manufacturer’s protocol. The quantitative
and qualitative analysis of extracted RNA was done using Nano-Drop® 2000 (Thermo Scientific,
Wilmington, DE). The RNA was then stored at -80ºC for further use in synthesis of cDNA.
cDNA Synthesis: Since 2-step RT-PCR method was being used, synthesis of cDNA from
the isolated RNA was carried out using the Verso cDNA Synthesis Kit (Thermo Scientific,
Waltham, MA) adhering to the manufacturer recommended method. RNA solution equivalent to
1 µg of RNA was used for cDNA synthesis. The synthesized cDNA was stored at -20 ºC till use in
RT-PCR.
RT-PCR Analysis: The amplification of gene of interest was carried out using gene specific
primers and Platinum® Pfx DNA Polymerase Kit (Life Technologies, Carlsbad, CA). The
manufacturer’s protocol was optimized while making the PCR reactions. Thermal Cycler (BioRad
Laboratories Inc., Hercules, CA) was used for running the PCR reactions. A gradient temperature
range was set up so that gene specific annealing temperatures could be employed while running
35
the cycles. A total of 40 cycles were run for each reaction with β-Actin being used as an internal
control. After amplification was achieved via thermal cycling, the PCR reactions were run on 1.2%
E-Gel® Agarose Gels containing SYBR Safe (Life Technologies, Carlsbad, CA) as per the
manufacturer’s recommendations using a 100bp Ladder. The gels were then visualized under
ChemiDoc XRS+ System (BioRad Laboratories Inc., Hercules, CA) using the preset protocol and
filters for visualization of gels containing SYBR Safe. The intensity of obtained bands was
measured using the ImageLab Software to achieve a semi-quantitative analysis of gene expression.
The sequence of primers used in the studies are as follows:
IL-10 Forward: CCAAGCCTTATCGGAAATGA
IL-10 Reverse: TCTCACCCAGGGAATTCAAA
TGF- β Forward: GAGCTGCGCTTGCAGAGATT
TGF- β Reverse: AGTTGGCATGGTAGCCCTTG
CCL-2 Forward: TCTCTCTTCCTCCACCACCAT
CCL-2 Reverse: CATTCCTTCTTGGGGTCAGCA
IL-1β Forward: GGCTGCTTCCAAACCTTTGA
IL-1β Reverse: GCTCATATGGGTCCGACAGC
TNF-α Forward: CATGAGCACAGAAAGCATGATC
TNF-α Reverse: CCTTCTCCAGCTGGAAGACT
IL-12a Forward: TGGATCTGAGCTGGACCCTT
IL-12a Reverse: TGGTCTTCAGCAGGTTTCGG
β-Actin Forward: GTTACCAACTGGGACGACA
β-Actin Reverse: TGGCCATCTCCTGCTCGAA
CSF-1R Forward: GACCTGCTCCACTTCTCCAG
CSF-1R Reverse: GGGTTCAGACCAAGCGAGAAG
36
GAPDH Forward: ACAGTCAGCCGCATCTTC
GAPDH Reverse: GCCCAATACGACCAAATCC
iNOS Forward: CCTTGGTGAAGGGACTGAGC
iNOS Reverse: TGCTGTGCTACAGTTCCGAG
Arg-1 Forward: AGCACTGAGGAAAGCTGGTC
Arg-1 Reverse: TACGTCTCGCAAGCCAATGT
4.1.3 Expression of Phenotype Specific Marker Genes by Flow Cytometry
The expression of surface markers of different macrophage phenotypes was determined by
use of antibodies directed against these markers. The anti-human antibodies were purchased from
Abcam (Cambridge, MA) and the anti-mouse antibodies were procured from Biolegend (San
Diego, CA). CD 86 and HLA-DR were used as markers for the M1 phenotype whereas CD 200R
and CD 163 were used as M2 markers. Ice cold PBS containing 10% FBS and 1% sodium azide
was used for harvesting, washing and resuspending cells. After the treatment with polarizing
stimulus, the media was removed and the cells were washed and harvested using PBS. The cells
were then centrifuged at 400g for 5 minutes to obtain a pellet. After decanting the PBS, the cells
were fixed to prevent internalization of surface markers by addition of 3 ml of 4% formalin solution
for 15 minutes. After centrifugation at 400g for another 5 minutes, the formalin solution was
discarded and the cells were washed thrice using PBS by centrifugation. The cells were then
incubated at room temperature in the dark for 1 hour with respective primary labeled antibodies.
Following incubation, the cells were washed thrice with PBS, centrifuged and re-suspended in 500
µl of PBS. The prepared samples were then analyzed on a FACSCalibur (BD Biosciences, San
Jose, CA) instrument using the BD CellQuest Pro software.
37
4.1.4 Preparation and Characterization of Nanoemulsions
The nanoemulsions were prepared using a two-step method described previously [77],
which comprised of high shear homogenization to make the coarse emulsion followed by high
energy ultrasonication to make the final nano-sized o/w emulsion. Briefly, Egg lecithin (120 mg)
and DSPE-PEG 2000 (15 mg) were added to a vial containing 4 ml of deionized water and stirred
for about half hour to prepare the aqueous phase. The oil phase was taken in a separate vial and
contained 1 ml of flaxseed, soy, safflower, or fish oil. The two phases were heated separately to
55-60ºC for 5 min. The aqueous phase was then added to the oil phase and homogenized at 9000
rpm for 5 minutes using a Silverson homogenizer (Silverson’s Model: L4RT-A; Silverson
Machines, East Longmeadow, MA) to make an O/W coarse emulsion. The coarse emulsion was
transferred to a new vial. The coarse emulsion was then subjected to ultrasonication at 40%
amplitude and 1 second pulse for 5 minutes. After sonication, the nanoemulsion was transferred
to a new vial to eliminate the larger size particles that may have stuck to the surface of the vial.
The nanoemulsions were then stored at 4ºC. The nanoemulsions were then characterized for size
and zeta potential using the Zetasizer Nano ZS90 (Malvern Instruments Ltd, Worcestershire,
United Kingdom).
4.2 Polarization Strategies
Various cell lines and stimulation protocol were attempted to investigate whether the
phenotype modulation can be attained. The various studies undertaken have been tabulated in
Table 3 below,
38
Table 3: Polarization approaches investigated in different cell lines
Cell Line Polarization
State
Concentration of
Polarizing Stimuli
Duration of
Stimuli
Polarization
Achieved
J774A.1 M1 LPS (100 ng/ml) 6 hours Yes
J774A.1 M2 Eicosapentanoic
Acid 24 hours No
J774A.1 M2 Resolvin D1 5 hours No
J774A.1 M1 and M2 Nanoemulsions 6, 12, 24 and 48
hours No
J774A.1 M2 Azithromycin (30
µM) 24 hours Yes
J77A.1 M2 IL-4 (100ng/ml) 16 hours Yes
RAW 264.7 M1 LPS (100 ng/ml) 6 hours No
THP-1 M1 LPS (100ng/ml
+IFN (20 ng/ml) 18 hours Partial
THP-1 M2 IL-4 (20ng/ml) 24 hours Partial
THP-1 M2 Azithromycin 24 hours Partial
39
4.2.1 Polarization of J77A.1 and RAW 264.7 macrophages using LPS
With the aim of promoting the classical or M1 phenotype of macrophages, the J774A.1
and RAW 264.7 cell line were stimulated with lipopolysaccharide (LPS) at a concentration of
100ng/ml for a period of 6 hours. After 6 hours the cells were harvested by scrapping them using
a cell scrapper. The expression of M1 and M2 specific marker genes at the mRNA level was then
ascertained using two-step RT-PCR.
4.2.2 Polarization of J77A.1 macrophages using Omega 3/6 Fatty Acid Nanoemulsion
Omega 3 and omega 6 fatty acids have been known to be associated with anti-inflammatory
and pro-inflammatory effects respectively. J774A.1 cells were treated with omega 3 and omega 6
fatty acid containing nanoemulsion formulation for 6, 12, 24 and 24 hours to ascertain if both M1
and M2 phenotypes can be induced. Two-step RT-PCR was performed to determine phenotypic
gene expression
4.2.3 Polarization of J77A.1 and RAW 264.7 macrophages using EPA and RvD1
The active components of the oils were then used for checking if the polarization could be
promoted. Since the M1 polarized phenotype was already established, emphasis was given on
induction of alternatively activated or M2 phenotype. Eicosapentanoic acid (EPA) and Resolvin
D1 (RvD1) were chosen for this purpose and purchased from Cayman Chemicals (Ann Arbor,
MI). EPA is an omega 3 fatty acid which has been shown to have anti-inflammatory effects. With
an aim to promote anti-inflammatory phenotype, J774A.1 cells were stimulated with 500 ng/ml of
EPA for a period of 24 hours. At the same time, the effect of RvD1, a precursor of another omega-
3 fatty acid docosahexanoic acid (DHA), on the macrophage phenotype at a concentration of 10nM
was investigated. Again 2-step RT-PCR was employed for determining gene expression.
40
4.2.4 Polarization of THP-1 Monocytes using LPS+ IFN and IL-4:
Since, significant M2 polarized phenotype was not achieved using the J774A.1 cell line, a
monocytic cell line THP-1 was used to check if phenotype modulation could be achieved. In order
to attain M1 and M2 polarized macrophages, THP-1 cells were stimulated with LPS (100 ng/ml)
plus IFN-gamma (20 ng/ml) and IL-4 (20 ng/ml) respectively for 6, 12, 18 and 24 hours. RT-PCR
and Flow cytometry. Analysis was then used to determine the expression at both mRNA and
protein level.
4.2.5 Polarization of THP-1 Monocytes using Azithromycin (AZM):
At the same time, during literature search showed that azithromycin (AZM) can also
induce polarization to M2 phenotype. With this information, the M2 polarizing capability of
AZM was tested by treating THP-1 cells with LPS+ IFN in the presence and absence of
azithromycin for 24 hours at both mRNA and protein level using 2-step RT-PCR and Flow
cytometry.
4.2.6 Polarization of J77A.1 macrophages using Azithromycin:
Based on preliminary data obtained with THP-1 cell line using AZM, the polarization
potential of azithromycin on J774A.1 cells was also evaluated. Similar procedure as discussed
under THP-1 polarization was used with J774A.1 cells. Again FACS and RT-PCR was used to
determine the expression profile of phenotypic markers.
4.2.7 Polarization of J77A.1 macrophages using IL-4:
J774A.1 macrophages were also treated with IL-4 at a concentration of 100ng/ml to
investigate if polarization to the M2 phenotype can be achieved. Reverse Transcriptase PCR was
then performed to analyze the expression of phenotype specific marker genes.
41
4.3 Formulation and Characterization of siRNA Encapsulating W/O/W Multiple Emulsion
4.3.1 Formulation of Blank and siRNA encapsulating W/O/W Multiple Emulsion: Water-in-
oil-in-water (W/O/W) multiple emulsion (ME) formulations that would encapsulate water soluble
payload were formulated using a 2 step emulsification method described previously [78] [79]. A
diagrammatic representation of this process is shown in Figure 20.
Figure 10: Diagrammatic representation of process of preparation of w/o/w ME
The safflower oil used in making the emulsion was kindly provided by Jedwards
International, Inc. (Braintree, MA). Span®80 and Pluronic® F-127 surfactants were purchased from
Sigma-Aldrich Inc. (St Louis, MO) and BASF Corporation (Mount Olive, NJ) respectively. The
ratio of Internal Aqueous: Oil: External aqueous phase was 1:2:3. Safflower oil-Span®80 Mixture
(9:1) was used to make primary emulsion with RNAse-free water by homogenization using
homogenizer (Silverson’s Model: L4RT-A; Silverson Machines, East Longmeadow, MA) at
42
10,000 rpm for 5 minutes. The primary emulsion thus formed was then re-emulsified with a 0.5%
w/v solution of Pluronic® F127 in RNAse free water by homogenization at 10,000 rpm for 15
minutes to create the W/O/W multiple emulsions. The emulsion was then stored at 4ºC and
characterized with respect to size, zeta potential. For preparing ME encapsulating scramble and
therapeutic siRNA, siRNA was purchased from SantaCruz Biotechnology Inc. (Dallas, TX) and
was resuspended in RNAse free water (Life Technologies, Grand Island, NY) to get a 20µM
solution. siRNA encapsulated multiple emulsion were prepared utilizing the same method as
described above except that siRNA was added to the internal aqueous phase. The prepared
emulsions were then evaluated for size, zeta potential as well as for siRNA stability and siRNA
encapsulation efficiency.
4.3.2 Size and Zeta Potential Measurement for Blank and siRNA Encapsulating ME: The
zeta potential of the multiple emulsion was determined using the Zetasizer Nano ZS90 (Malvern
Instruments Ltd, Worcestershire, United Kingdom). The emulsion was diluted 1000 fold using
RNAse free water containing 0.5% w/v solution of Pluronic® F127 before the measurements were
carried out. The size of the oil droplets were determined manually by measuring the size using a
micrometer screw gauge. Five optical fields each containing 25 oil droplets were viewed for
determining the size using 40x magnification.
4.3.3 Qualitative Determination of siRNA Encapsulation by ME: In order to determine if the
emulsion system can encapsulate molecules into the internal aqueous phase, a water soluble dye
tetramethyl rhodamine dextran (TMR-D) was for encapsulation. After preparing the emulsion, a
sample was checked under a fluorescence microscope to ascertain if encapsulation of TMR-D was
achieved.
43
4.3.4 Quantitative Determination of siRNA Encapsulation by ME: The encapsulation
efficiency of the ME system was determined quantitatively using the Picogreen Assay. The Quant-
iT™ PicoGreen® dsDNA Reagent was purchased from Life Technologies (Carlsbad, CA). The
procedure provided by the manufacturer was modified for adaption to a 96 well format. A standard
curve was obtained by preparing different concentrations of the siRNA in RNAse free water and
reading the fluorescence after incubation with PicoGreen reagent for 5 minutes using the Synergy®
HT microplate reader (Bio-Tek Inc., Winooski, VT) supplemented with the KC4 software. The
external aqueous phase was separated by centrifuging emulsion at 13,000 rpm using the Amicon®
Ultra – 0.5ml centrifugal filter (Merck Millipore Ltd, Carrigtwohill, Ireland). The external phase
was then freeze dried using Freeze Dryer (Labonco Corporation, Kansas City, MO) to concentrate
the free siRNA that may be present.
After freeze drying the mass left in the centrifuge tube was re-suspended in 230 µl of
RNAse free water. The percent encapsulation was then determined using the following formula:
% Encapsulation = (Free siRNA in the external Phase / Total siRNA loaded) x 100
Also to confirm the absence of siRNA in the external phase, the above concentrated external phase
was run on an E-Gel 4% EX Agarose Gel (Life Technologies, Carlsbad, CA) using an ultra-low
range ladder (Thermo Fisher, Waltham, MA) and the encapsulated siRNA as a control.
4.3.5 Determination of Stability of Encapsulated siRNA: In order to assess the stability of
encapsulated siRNA, the siRNA was extracted using a liquid-liquid extraction technique
employing chloroform (CHCl3) and Isopropyl alcohol (IPA). The extraction method was
optimized such that the ratio of emulsion: CHCl3: IPA was 1:5:2. The oil globules encapsulating
the siRNA were solubilized in chloroform. Isopropyl alcohol precipitated the siRNA which
selectively partitioned into the aqueous phase. The above mixture was centrifuged at 13,000 rpm
44
for 30 minutes. 2 layers for obtained after centrifugation, an upper aqueous layer and a lower
CHCl3-IPA layer. The aqueous layer at the top was collected using a pipette. The collected aqueous
phase was then diluted in different ratios and then run on a 4% Agarose gel. The gel run was chosen
as per manufacturer instructions and was 15 minutes. Free siRNA was also run on the same gel
and served as a control.
4.4 Gene Silencing and Cellular Uptake of ME encapsulated CSF-1R siRNA
With the aim of evaluating the potential and extent of silencing induced by CSF-1R
siRNA, the J774A.1 macrophages were plated in a 6 well plate at a density of 100,000 cells per
well and allowed to grow overnight. The next day, the cells were treated with ME encapsulated
CSF-1R siRNA for 6 hours at 37oC. Thereafter the cells were washed with PBS and then
incubated for further 24 or 48 hours. Untreated cells, naked siRNA, Lipofectamine-siRNA
complex, blank ME and scrambled siRNA encapsulated in ME served as controls. Lipofectamine
RNAiMax (Life Technologies, Carlsbad, CA) for making the siRNA-Lipofectamine complex by
following the manufacturer recommended protocol. After the incubations for aforementioned
time, the cells were harvested and the RNA isolation and cDNA synthesis was performed as per
the method described in Section 4.1.2. The expression of CSF-1R was then determined by
quantitative real-time PCR (qPCR) using the ∆∆Ct Method. The LightCycler® 480 SYBR Green
I Master Mix (Roche Diagnostics, Indianapolis, IN) was used for preparing various reactions and
Beta-Actin was used as an endogenous control. The Roche Light Cycle 480 Instrument was used
for running the reactions.
In order to determine if the siRNA encapsulated in the multiple emulsion was taken up by
the J77A.1 macrophages, cell uptake studies were carried out using ME encapsulating Alexa647
labelled siRNA (Qiagen, Valencia). Alcohol sterilized cover slip were placed in each well of a 6-
45
well plate and kept in UV light for 20-30 minutes. Thereafter approximately 200, 000 cells were
plated and allowed to adhere overnight before treatment with test and control formulations could
be started the next day. The cells will be incubated with these formulations for 60, 120 and 240
minutes respectively with Hoechst being added to culture media in the recommended 15 min
before the coverslips will be withdrawn from the 6 well plates. Following incubation, the cells
will be washed with sterile ice cold PBS and fixed with 3.6% w/v formaldehyde for 15 minutes.
The coverslips will then be removed and placed on a clean slide on which a drop of Immuno-
Mount (Richard Allen Scientific, Kalamazoo, MI) will be added. The slides will then be
visualized under a LSM 700 Confocal microscope with Z-stack capabilities.
4.5 Amplification, Purification, Isolation and Characterization of Plasmid DNA
4.5.1 Amplification, Purification and Isolation of Plasmid DNA:
The bacteria transformed with plasmids encoding miR-155 and Null plasmid (Plasmid without the
miR-155 sequence) were purchased from Cell BioLabs (San Diego, CA) LB Media and LB Agar
Media was prepared by dissolving 25gm of the powdered LB media (Fisher BioReagents, Fair
Lawn, NJ) and 25gm of powdered media plus 15gm of Agar (Fisher Scientific, Pittsburg, PA) in
water to make 1L of LB and LB Agar Media respectively. The media were then autoclaved for 30
minutes. Once the media cooled to below 55oC, ampicillin was added to the media at a final
concentration of 10µg/ml and the bottle was inverted a couple of times to ensure uniform mixing
of the antibiotic. The LB Agar Media was then poured into petri dishes and stored at 4oC. The
QIAFilter Mega Kit (Qiagen, Valencia) was used for the purification and isolation of plasmid
DNA. The amplification, purification and isolation of the plasmid was conducted as per the scheme
shown below,
46
Figure 11: Methodology for Plasmid Amplification, Purification and Isolation
4.5.2 Characterization of Plasmid DNA:
In order to ascertain that the correct plasmids were purified and isolated and also to determine the
presence of the miR-155 insert, Agarose Gel Electrophoresis was employed. The isolated plasmids
were run on a gel with the miR-155 insert size being examined by restriction enzyme digestion
followed by electrophoresis using 1% E-Gel® EX Agarose Gels (Life Technologies, Kiryat
Shmona, Israel) BamHI and NheI restriction enzymes were purchased from New England BioLabs
(Ipswich, MA) and the manufacturer recommended protocol was used for the digestion reaction.
Digestion with single enzyme and digestion with both enzymes was also performed to demonstrate
specificity of enzyme and stability of plasmids.
47
4.6 Formulation and Characterization of plasmid encapsulating W/O/W Multiple Emulsion
4.6.1 Formulation of Blank and Plasmid encapsulating W/O/W ME:
The formulation of Blank ME (BME) was same as described in section 4.3.1. When formulating
ME encapsulating null plasmid (NME) or miR-155 encoding plasmid (MME), 500 µl of respective
plasmids (7000 ng/µl) in RNAse free water were used in place of internal phase to prepare 3ml of
ME.
4.6.2 Size and Zeta Potential Measurement for Blank, Null Plasmid and miR-155 Plasmid
encapsulating ME:
The size and zeta potential measurements were carried out by following the same method as
described in Section 4.3.2
4.6.3 Quantitative Determination of Plasmid DNA Encapsulation:
In order to quantitate the encapsulation efficiency of the ME system, the amount of plasmid present
in the internal aqueous and the external aqueous phase was determined using the Picogreen Assay.
50 µl of the null plasmid and miR-155 plasmid encapsulated ME was taken. The external aqueous
phase was separated by centrifuging emulsion at 13,000 rpm using the Amicon® Ultra – 0.5ml
centrifugal filter (Merck Millipore, Billerica, MA) for 30 minutes. The retained primary W/O
emulsion was then dissolved in Isopropyl Alcohol (IPA) to extract and precipitate the plasmid
DNA which on centrifugation at 13,000 rpm for 30 minutes formed a pellet. The supernatant was
discarder and the pellet was suspended in 120 µl of RNAse free water. 1 µl of this was used in the
PicoGreen Assay. The Quant-iT™ PicoGreen® dsDNA Reagent was purchased from Life
Technologies (Carlsbad, CA). The procedure provided by the manufacturer was modified for
adaption to a 96 well format. A standard curve was obtained by preparing different concentrations
48
of the plasmid DNA in RNAse free water and reading the fluorescence after incubation with
PicoGreen reagent for 5 minutes using the Synergy® HT microplate reader (Bio-Tek Inc.,
Winooski, VT) supplemented with the KC4 software. The amount of plasmid present in the
internal and external aqueous phase was then determined using the obtained standard curve. The
percent encapsulation was then determined using the following formula,
4.6.4 Stability Determination of Encapsulated Plasmid & DNAse Protection Assay
This study was designed to ascertain the stability and integrity of the encapsulated plasmid as well
as evaluate the ability of formulation to protect the encapsulated DNA plasmid against DNAse
action. In order to test this, 50 µl of the ME encapsulating plasmid was either left untreated or
treated with DNAse for 37oC for 20 minutes. Thereafter the untreated emulsion and the DNAse
treated emulsion were centrifuged at 13,000 rpm for 30 minutes to separate the external aqueous
phase as well as get rid of the DNAse present. The internal phase was then isolated as per the
method described in Section 4.5.3 and ran on a 0.8% Agarose Gel. The null and the miRNA
encoding plasmids with and without DNAse treatment were also run on the same gels and served
as controls. The external phase was also run on the gel to demonstrate the absence of plasmid in
the external phase.
4.7 Cellular Uptake
In order to investigate the uptake of miRNA encoding plasmid by J774A.1 macrophages,
3.5mg of plasmid DNA was labelled with 500 µl of 1 mg/ml solution of Propidium Iodide (Sigma
Aldrich, ). The labeled plasmid DNA was then encapsulated in the internal aqueous phase of the
ME. Lab-Tek 4 chamber slides (Thermo Scientific, Rochester, NY) were kept in UV light for 20-
30 minutes. Thereafter approximately 200, 000 cells were plated and allowed to adhere overnight
% Encapsulation = (Plasmid DNA in the Internal Phase / Total Plasmid DNA loaded) x 100
49
before treatment with different conditions was started the next day. The cells were left untreated
or treated with naked plasmid, BME or MME and incubated for 60,120, 240 and 360 minutes
respectively, with Hoechst 33342 (Life Technologies, Eugene, Oregon) being added to culture
media in the recommended concentrations 15 min before the end of the incubation period.
Following incubation, the cells were washed with sterile ice cold PBS and fixed with 3.6% w/v
formaldehyde for 15 minutes. The chambers were then removed following the manufacturer
instructions and a clean cover slip was then placed on the slide on which a drop of Immuno-Mount
(Richard Allen Scientific, Kalamazoo, MI) was added. The slides were then visualized under a
LSM 700 Confocal microscope (Carl Zeiss Microscopy) with Z-stack capabilities.
4.8 Quantitative Determination of Intracellular miR-155 Production
The main goal of this study was to ascertain the intracellular levels of miR-155 attained
after various treatments. 200,000 cells were plated in 6 well plates and allowed to grow overnight.
The following day the cells were either left untreated or treated with either naked plasmid (20 µg)
or Lipofectamine3000-Plasmid Complex or blank ME (BME), null plasmid vector encapsulated
ME (NME) or microRNA-155 plasmid encapsulated ME (MME) for 4 hours. Thereafter cells were
washed thrice with PBS and then allowed to grow for 24, 48, 72, 96 and 120 hours. At each time
point cells were harvested and the RNA was extracted using the Quick-RNA™ MiniPrep Kit
(Zymo Research Corp., Irvine, CA). The isolated RNA was analyzed quantitatively and
qualitatively using Nano-Drop® 2000 (Thermo Scientific, Wilmington, DE). 100 ng of the RNA
was reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit (Life
Technologies, Foster City, CA) and the recommended protocol. The cDNA obtained was then used
in setting up qPCR reactions according to the recommended protocol using the TaqMan probes for
mature mmu-miR155-3p (Life Technologies, Foster City, CA) and for snoRNA202 (Life
50
Technologies, Foster City, CA) which was used as an endogenous control. The ∆∆Ct Method was
then used for determining the expression levels of miR155 and snoRNA202.
4.9 Gene Expression & Macrophage Re-polarization Analysis
The aim of this study was to determine the efficacy of miR-155 encoding plasmid in
repolarizing macrophages from M2 to M1 phenotypic state. Approximately 200,000 cells were
plated in a T-25 flask and allowed to grow overnight. The following day the cells were left
untreated or incubated with naked plasmid, plasmid-lipofectamine complex, BME, NME and
MME for 4 hours. Lipofectamine 3000 (Life Technologies, was used for making the complexes
by following the manufacturer recommended protocol. Thereafter the cells were washed twice
with PBS to remove the residual oil that remained stuck to the plastic. The cells were then
incubated and allowed to grow for further 44 hours to enable the production of miR-155. Sixteen
hours before the end of this 48 hour incubation period, cells were stimulated with 100ng/ml IL-4
to induce their polarization to M2 phenotype. The effect of miRNA 155 on the expression of M1
and M2 phenotype specific marker genes was then assessed using qPCR. The time points chosen
were 12 hours, 24 hours and 48 hours post IL-4 stimulation. The cells were harvested after the said
time points and the RNA was extracted and cDNA was synthesized as described in Section 4.1.2.
The LightCycler® 480 SYBR Green I Master Mix (Roche Diagnostics, Indianapolis, IN) was used
for preparing various reactions for qPCR and the manufacturer recommended protocol was
followed while making the reactions and running the plate. GAPDH was used as an endogenous
control. The Roche Light Cycle 480 Instrument was used for running the reactions. The ∆∆Ct
Method was used for determining the expression levels of various genes under various treatment
conditions.
51
4.10 Cytotoxicity Analysis
The objective of this study was to ascertain the toxicity of the multiple emulsion
formulation in J774A.1 macrophages. The CellTiter 96® AQueous One Solution Cell Proliferation
Assay (MTS Assay) (Promega, Madison, WI) was used to determine the cytotoxicity of miRNA-
155 plasmid encapsulated multiple emulsion. The scheme of the study is as shown below,
The absorbance was read at 490nm using the Synergy HT Plate reader (BioTek Inc., Winooski,
VT) and the percent cell viability was determined using the following formula,
% Cell Viability = ����������� ����/���������������������� x 100
Figure 12: Experimental Scheme for Cytotoxicity Study
52
5. RESULTS AND DISCUSSION
5.1 Macrophage Polarization Strategies
5.1.1 Polarization of J77A.1 and RAW 264.7 macrophages using LPS
From the Figure 13 below, it can be clearly seen that the expression of M1 markers (IL-
1β and TNF-α) was increased compared to the control (untreated J774A.1 cells) whereas the
levels of M2 markers (TGF-β and IL-10) were downregulated. A simultaneous increase in
expression of M1 markers and decrease in M2 markers was indicative of polarization of
macrophages to the M1 phenotype. Since the results for J774A.1 were better in comparison to
RAW 264.7, it was decided that J77A.1 cell line will be used for further polarization
experiments.
Figure 13: Semi-quantitative analysis of M1 and M2 specific gene expression after 6
hour LPS stimulation (100 ng/ml)
5.1.2 Polarization of J77A.1 macrophages using Omega 3/6 Fatty Acid Nanoemulsion
Figure 14 and 15 below demonstrate that neither of the emulsion could significantly
change the expression of phenotypic markers.
9.19
33.9926.53
14.96
79.86
102.62
0.6710.48
0
20
40
60
80
100
120
IL-1 TNF TGF IL-10
% R
ela
tiv
e E
xp
ress
ion
no
rma
lize
d t
o β
-Act
in
Gene
Relative Expression of M1 and M2 Specific Genes in response to 6 hour LPS
stimulation (100 ng/ml)
Unstimulated
6 Hour Stimulated
M1 Markers M2 Markers
53
Figure 14: Semi-quantitative analysis of M1 and M2 specific gene expression after 6 and 12
hour treatment with different nanoemulsions
Figure 15: Semi-quantitative analysis of M1 and M2 specific gene expression after 24 and
48 hour treatment with different nanoemulsions
13
101
23
56
176
220 1
251 6
291
3617
3 531
0
50
100
150
200
IL-1 TNF TGF
% R
ela
tiv
e
Ex
pre
ssio
n
no
rma
lize
d t
o β
-
Act
in
Gene
Relative Expression of M1 and M2 Specific Genes after 6 Hour Treatment with
various Nanoemulsions
Unstimulated
6 Hr LPS Stimulated
6 Hr Soy Oil NE
6 Hr Flaxseed Oil NE
6 Hr Safflower oil NE
6 Hr Fish Oil NE
13
101
23
56
176
220 0 03 3 101 1 43
3315
0
50
100
150
200
IL-1 TNF TGF
% R
ela
tiv
e
Ex
pre
ssio
n
no
rma
lize
d t
o β
-
Act
in
Gene
Relative Expression of M1 and M2 Specific Genes after 12 Hour Treatment with
various NanoemulsionsUnstimulated
6 Hr LPS Stimulated
12 Hr Soy Oil NE
12 Hr Flaxseed Oil NE
12 Hr Safflower oil NE
12 Hr Fish Oil NE
54
5.1.3 Polarization of J77A.1 and RAW 264.7 macrophages using EPA and RvD1
As can be seen from Figure 16 below, the expression of M1 phenotypic markers decreased
after treatment with both EPA and RvD1 however the levels of M2 markers also decreased in
comparison to either unstimulated or the LPS stimulated cells. Since the study was conducted at a
single time point, the change in expression pattern could not be investigated on account of the
differential kinetics of expression of various markers.
Figure 16: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression
after treatment with EPA and RvD1
5.1.4 Polarization of THP-1 Monocytes using LPS+ IFN and IL-4
Favorable results were obtained at 18 hour for M1 markers (CCR7, CXCL 11) and at 24
hour for M2 markers (SR-B1, TGF-β, CCL22) using RT-PCR, as shown in Figure 17 and 18.
Based on these findings, FACS was done at these time points. FACS data shown in Figure 19
revealed that a down-regulation in M1 markers was seen at 18 however the expression of M2
markers did not show any increase.
16
131
93
23
67
134
86
2614
104
68
16
57
102
45
21
0
50
100
150
IL-1 TNF TGF IL-10
% R
ela
tiv
e E
xp
ress
ion
no
rma
lize
d t
o
β-A
ctin
Gene
Relative Expression of M1 and M2 Specific Genes after treatment with EPA
or RvD1 Unstimulated
6 Hr. LPS Treated
24 Hr. EPA Treated
6 Hr. LPS + 5 Hr. RvD1
Treated
55
Figure 17: Semi-quantitative analysis of M1 phenotype specific gene expression after M1
and M2 polarizing stimuli in THP-1 cells
Figure 18: Semi-quantitative analysis of M2 phenotype specific gene expression after M1
and M2 polarizing stimuli in THP-1 cells
Figure 19: Expression of Surface markers of M1 and M2 phenotypes after M1 and M2
polarizing stimuli in THP-1 cells
17
31
59 61
2531
0
20
40
60
80
CCR7 CXCL11Pe
rce
nt
Re
lati
ve
Ex
pre
ssio
n
no
rma
lize
d t
o B
eta
-Act
in
Genes
Expression of M1 Marker Genes after 18 hour treatment with M1 and
M2 polarizing Stimuli
Untreated 18 Hr. M1 Polarized 18 Hr. M2 Polarized
140
83
60
130
9888
169
132
96
0
20
40
60
80
100
120
140
160
180
SRB-1 TGF CCL22
Pe
rce
nt
Re
lati
ve
Ex
pre
ssio
n
no
rma
lize
d t
o B
eta
-Act
in
Genes
Expression of M2 Marker Genes after 24 hour treatment with M1 and M2
polarizing StimuliUntreated 24 Hr. M1 Polarized 24 Hr. M2 Polarized
15.911.55
37.5
1413.116.85
0
10
20
30
40
CD 80 [FL-3] CD 200R [FL-2]
MF
I
Surface Markers
Surface Marker Expression in 18 hours after Various TreatmentsUntreated M1 Polarized M2 Polarized
56
5.1.5 Polarization of THP-1 Monocytes using Azithromycin (AZM)
Figure 20 indicates that treatment with azithromycin decreased the level of M1 markers (CCR7
and CXCL11) and simultaneously increased the expression of M2 marker (TGF).
Figure 20: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression
after LPS and IFN treatment in the presence and absence of AZM in THP-1 cells
5.1.6 Polarization of J77A.1 macrophages using Azithromycin
Figure 21: Expression of Surface markers of M1 and M2 phenotypes after AZM treatment
in J774A.1 cells
As is evident from the Figure 21, a decrease in M1 marker (CD 80) and marginal increase
in M2 markers (CD 200R) demonstrates a shift in the phenotypic population from M1 to M2 in
the presence of azithromycin. These results using flow cytometry were corroborated by RT-PCR
results shown in Figure 22. The levels of M2 marker CCL2 slightly increased in the presence of
10
54
79
7 5
8291 91
768172
99
0
50
100
150
CCR7 CXCL 11 TGF
Pe
rce
nt
Re
lati
ve
Ex
pre
ssio
n n
orm
ali
zed
to B
eta
-Act
in
Genes
Expression of M1 and M2 Marker Genes 24 hourd after various
TreatmentsUntreated Azithromycin Only LPS + IFN LPS +IFN + Azithromycin
5.294.55
5.88 5.57
11.08
6.747.56 7.56
0
5
10
15
CD 80 (FL 1) CD 200R (FL 2)
MF
I
Surface Markers
Surface Marker Expression 24 hours after various treatments
Untreated 30 uM Azith. LPS+IFN LPS+IFN+30 uM Azith.
57
azithromycin with a simultaneous decrease in expression of M1 markers (TNF-α and IL-12a). Thus
the data from both FACS and PCR clearly indicate that the phenotypic modulation of macrophages
can be achieved under different stimulating conditions.
Figure 22: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression
after LPS and IFN treatment in the presence and absence of AZM in J774A.1 cells
5.1.7 Polarization of J77A.1 macrophages using IL-4:
Figure 23: Semi-quantitative analysis of M1 and M2 phenotype specific gene expression
after 16h IL-4 (100ng/ml) Stimulation in J774A.1 cells
Figure 23 below reveals that IL-4 stimulation for 16 hours causes the M2 markers IL-10 and
Arg-1 to increase significantly with a simultaneous decrease in M1 marker (iNOS). This result is
indicative of polarization of macrophages to a M2 phenotype.
34%
99%
34%42%
91%
48%
71%
184%
47%32%
102%
78%
0%
50%
100%
150%
200%
IL-12a TNF α CCL 2Pe
rce
nt
Re
lati
ve
Ex
pre
ssio
n
no
rma
lize
d t
o B
eta
-Act
in
Gene
Expression of M1 and M2 Marker Genes 24 hours after various Treatments
Untreated 30 uM AZM LPS + IFN LPS +IFN + 30 uM AZM
22.31
3.24 6.46 8.6012.303.20
98.37
81.30
0
50
100
150
iNOS TNF-alpha Arg-1 IL-10
% R
ela
tiv
e E
xp
ress
ion
no
rma
lize
d t
o β
-Act
in
Gene
Relative Expression of M1 and M2 Specific Genes in response to 16 hour
IL-4 Stimulation (100 ng/ml)
Untreated IL-4 Stimulated
58
5.2 Characterization of Blank and siRNA encapsulating W/O/W Multiple Emulsion
5.2.1 2 Size and Zeta Potential Measurement for Blank and siRNA encapsulating ME
The size of the oil droplets as seen from Table 4 was around 0.8 µ which is similar to size
of most pathogenic organisms and thus ideal for recognition by macrophages.
Table 4: Size and Zeta Potential Measurements of Blank and siRNA encapsulated ME
Sample Size (µm) Zeta (mV)
Blank ME 0.89 ± 0.22 -46.5 ± 4.79
siRNA encapsulated ME 0.8 ± .20 -47.1 ± 3.05
5.2.2 Qualitative Determination of Encapsulation by ME
The image in Figure 24 clearly shows that the system can encapsulate water soluble
payload which does not leak into the surrounding external phase.
Figure 24: Bright-field image showing oil globules having diameter of 1.0 um or less and
Fluorescent Microscopy image of a stable non-leaky ME encapsulating dextran conjugated
tetramethyl-rhodamine. (Magnification: 40X)
59
5.2.3 Quantitative Determination of siRNA Encapsulation by ME
The siRNA encapsulation efficiency calculated using the Standard Curve below was 99.7%.
Figure 25: Standard Curve for PicoGreen Assay for siRNA Encapsulation
Additionally, as is evident from the agarose gel electrophoresis image in Figure 26 below,
siRNA was not detected in the external aqueous phase of multiple emulsion.
Figure 26: Agarose Gel Image showing absence of siRNA in external aqueous phase of
W/O/W/ multiple emulsion
5.2.4 Determination of Stability of Encapsulated siRNA
Figure 27 demonstrates that the siRNA was not affected during the formulation process and
maintained stability after encapsulation in the emulsion system
y = 16958x + 191.18
R2 = 0.992
-10000
0
10000
20000
30000
40000
50000
0 0.5 1 1.5 2 2.5 3
Flu
ore
sce
nce
(R
FU
)
Concentration (ng/µl)
PicoGreen Assay Standard Curve
60
5.3 Gene Silencing and Cellular Uptake of ME encapsulated CSF-1R siRNA
Figure 27: Agarose Gel Image showing the stability of extracted siRNA
100.00 91.59100.70 104.49 109.43
90.33
0
50
100
150
Untreated Naked siRNA 24hScramble 24h 25 nM 24h 50 nM 24h 100 nM 24h% R
ela
tive
Exp
ress
ion
no
rma
lize
d t
o β
-Act
in
Treatments
Expression of CSF-1R 24 hours post various treatments
100.00
135.03
76.49
96.82
68.7856.64
0
50
100
150
Untreated Naked siRNA 48h Scramble 48h 25 nM 48h 50 nM 48h 100 nM 48h
% R
ela
tive
Exp
ress
ion
no
rma
lize
d t
o β
-Act
in
Treatments
Expression of CSF-1R 48 hours post various treatments
Figure 28: Quantitative Analysis of CSF-1R expression 24 & 48 hour after treatment with
different concentrations of siRNA
61
Figure 28 demonstrated that the CSF-1R siRNA was capable of silencing CSF-1R expression in
a dose dependent manner 48 hour post siRNA treatment with 100nM concentration showing
44% silencing.
From Figure 29 it can be interpreted that the multiple emulsion encapsulating the CSF-1R
siRNA was not capable of down regulating the CSF-1R expression.
From Figure 30 it can be confirmed that the multiple emulsion system encapsulating the siRNA
was capable of delivering the siRNA to the cells which was taken up as early as 1 hour after
incubation.
100.00
142.41
10.01
81.98
116.20103.05
-50
0
50
100
150
200
Untreated Naked siRNA
24h
Lipofectamine
+ siRNA 24h
Blank ME 24h Scramble ME
24h
CSF-1R ME 24h
% R
ela
tive
Exp
ress
ion
no
rma
lize
d t
o β
-Act
in
Treatments
Expression of CSF-1R 24 hours post various treatments
100.00 108.92
162.45185.75
155.83
121.70
0
50
100
150
200
250
Untreated Naked siRNA
48h
Lipofectamine
+ siRNA 48h
Blank ME 48h Scramble ME
48h
CSF-1R ME 48h
% R
ela
tive
Exp
ress
ion
no
rma
lize
d t
o β
-Act
in
Treatments
Expression of CSF-1R 48 hours post various treatments
Figure 29: Quantitative CSF-1R Expression Analysis 24 and 48 hours post various treatments
62
1
Hour
2
Hours
4
Hours
Figure 30: Cellular Uptake of Alexa 647 labelled siRNA encapsulated in multiple emulsion
post 1, 2 and 4 hours of incubation in J774A.1 macrophages (Magnification: 40x)
63
5.4 Characterization of Plasmid DNA
Figure 31: Figure 31: (A & B) Null and miR-155 encoding expression plasmid size and
features (C) miR-155 encoding insert with restriction site sequence for BamHI and NheI
As seen in Figure 32, the intact null and miR-155 encoding plasmid demonstrated a size of
around 4.7kb and 5kb respectively. However, the single enzyme and double enzyme digested
demonstrated a size above 4.7kb and 5kb respectively because of linearization. The null plasmid
after digestion did not show presence of fragment whereas the mir-155 encoding plasmid
displayed an insert size of 314bp which was in accordance with that declared by the
manufacturer.
64
Figure 32: Agarose Gel Image showing the size of Null and miR-155 Plasmid and miR-155
insert after restriction Digestion
5.5 Characterization of Plasmid encapsulated W/O/W Multiple Emulsion
5.5.1 Size and Zeta Potential Measurement for Blank, Null Plasmid and miR-155 Plasmid
encapsulating ME
The size of oil droplets in the different types of emulsions were about 0.9 microns and the zeta
potential was in the range of -46 to -47mV.
Table 5: Size and Zeta Potential for Blank, Null Plasmid and miR-155 Plasmid
encapsulating ME
65
5.5.2 Quantitative Determination of Plasmid DNA Encapsulation
The standard curve obtained for the PicoGreen Assay is as shown below.
Figure 33: Standard Curve for PicoGreen Assay for Plasmid Encapsulation
The encapsulation efficiency calculated using the above standard curve revealed that miR-155
encoding plasmid and null plasmid encapsulating ME showed about 60 and 65% encapsulation
respectively.
Table 6: Encapsulation Efficiency of mir-155 and Null Plasmid ME
66
5.5.3 Stability Determination of Encapsulated Plasmid & DNAse Protection Assay
Figure 34 above clearly showed that both the null plasmid and the miR-155 encoding plasmid that
was encapsulated in the multiple emulsion system maintained its stability and integrity after the
formulation process. Also the encapsulated plasmid was not degraded after the multiple emulsion
was treated with DNAse. Additionally, the image displayed the absence of plasmid in the external
phase of the multiple emulsion.
5.6 Cellular Uptake
The microscopy images in Figure 35 demonstrated that the multiple emulsion system
encapsulating plasmid was rapidly taken up by J774A.1 macrophages starting as early as 1 hour
after incubation. The uptake increased as the time progressed and maximum uptake as evident
from fluorescence signal was observed at the 6 hour post-incubation.
Figure 34: Agarose Gel Image showing stability and protection against DNAse treatment of
encapsulated Plasmid
67
1 Hour 1 Hour 2 Hour 4 Hour 6 Hour
Un
treated
N
ak
ed P
lasm
id
BM
E
MM
E
Figure 35: Fluorescence and brightfield images showing uptake of multiple emulsion encapsulating
plasmid 1, 2, 4 and 6 hour post incubation.(Magnification: 40x)
68
5.7 Quantitative Determination of Intracellular miR-155 Production
From the graph in Figure 36 it can be comprehended that there was a time dependent increase in
the intracellular levels of miR-155 after treatment with multiple emulsion encapsulating plasmid.
The intracellular expression started around 48 hours, reached maximum by 72 hours and then
declined. With respect to lipofectamine, after the initial increase in intracellular expression at
24h, the expression remained fairly constant up to 96 hours and then showed a drastic decline.
Figure 36: Quantitative Analysis of Intracellular miR-155 levels under various conditions
5.8 Gene Expression & Macrophage Re-polarization Analysis
It was concluded from Figure 37 and 38 that levels of pro-inflammatory M1 marker IL-1β and
iNOS increased 6 and 2.5 fold respectively after lipofectamine and 12 and 3 fold respectively for
multiple emulsion treatment at 48 hours post IL-4 stimulation which coincided with the time
frame at which maximum intracellular expression of miR-155 was achieved.
1.00
8.34
0.33
3.89
1.04
4.21
2.93
4.01
1.512.10
1.41
0
1
2
3
4
5
6
7
8
9
10
Re
lati
ve
Ex
pre
ssio
n m
iR1
55
/sn
o 2
02
Condition
Intracellular miR-155 Expression
69
Figure 37: Quantitative Determination of IL-1β expression after various treatments 12, 24
and 48 hours post IL-4 Stimulation
1.00
0.64
0.23
0.49
0.69
0.58
0.85
0.00
0.20
0.40
0.60
0.80
1.00
1.20IL
-1β
Ex
pre
ssio
n
no
rma
lize
d t
o G
AP
DH
Conditions
IL-1β Expression 12h post IL-4 Stimulation after various
treatments
1.00
0.39
0.21
0.52
0.36 0.32
0.85
0.00
0.20
0.40
0.60
0.80
1.00
1.20
IL-1
βE
xp
ress
ion
no
rma
lize
d
to G
AP
DH
Conditions
IL-1β Expression 24h post IL-4 Stimulation after various
treatments
1.00 1.39
6.26
1.07 1.59
12.76
0.85
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
IL-1
βE
xp
ress
ion
no
rma
lize
d
to G
AP
DH
Conditions
IL-1β Expression 48h post IL-4 Stimulation after various
treatments
70
Figure 38: Quantitative Determination of iNOS expression after various treatments 12, 24
and 48 hours post IL-4 Stimulation
1.220.98
0.350.50
0.18 0.23
0.72
0.00
0.50
1.00
1.50
2.00
2.50
iNO
S E
xp
ress
ion
no
rma
lize
d t
o G
AP
DH
Conditions
iNOS Expression 12h post IL-4 Stimulation after various
treatments
1.011.17
0.98
1.27
0.21
1.37
0.72
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
iNO
S E
xp
ress
ion
no
rma
lize
d t
o G
AP
DH
Conditions
iNOS Expression 24h post IL-4 Stimulation after various
treatments
1.010.79
2.48
1.471.29
3.05
0.72
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
iNO
S E
xp
ress
ion
no
rma
lize
d t
o G
AP
DH
Conditions
iNOS Expression 48h post IL-4 Stimulation after various
treatments
71
Figure 39: Quantitative Determination of iNOS expression after various treatments 12, 24
and 48 hours post IL-4 Stimulation
1
3,060
14,040
4,8833,383
1,241
10,205
0.00
4000.00
8000.00
12000.00
16000.00A
rg-1
Ex
pre
ssio
n
no
rma
lize
d t
o G
AP
DH
Conditions
Arg-1 Expression 12h post IL-4 Stimulation after various
treatments
1.01
18396.29
4289.55 4094.20 3956.566580.74
10205.40
0.00
5000.00
10000.00
15000.00
20000.00
25000.00
Arg
-1 E
xp
ress
ion
no
rma
lize
d t
o G
AP
DH
Conditions
Arg-1 Expression 24h post IL-4 Stimulation after various
treatments
1
3,601
802 1,365 1,545
4,465
10,205
0.00
5000.00
10000.00
15000.00
Arg
-1 E
xp
ress
ion
no
rma
lize
d t
o G
AP
DH
Conditions
Arg-1 Expression 48h post IL-4 Stimulation after various
treatments
72
Figure 39 indicated that there was a decrease in expression levels of Arg-1 but this was seen
across different treatments and could be due to the natural kinetics of Arg-1 rather than an effect
caused by intracellular production of miR-155.
5.9 Cytotoxicity Analysis
Figure 40 proved that the multiple emulsion system were tolerated by the J774A.1 macrophages
showing 80-90% viability 24 and 48 post treatment. The positive control PEI on the other hand
was toxic and resulted in 14.7 and 6.8% viability at similar time points.
Figure 40: Cytotoxicity Analysis of ME System in J77A.1 macrophages 24 and 48h after
treatment
100.00 98.2191.79 90.77 89.79 90.20
14.76
0
20
40
60
80
100
120
Pe
rce
nt
Ce
ll V
iab
ilit
y
Condition
Percent Cell Viability under various conditions 24 hours post treatment
100.00 99.8491.69
86.73 87.67 87.45
6.87
0
20
40
60
80
100
120
Pe
rce
nt
Ce
ll V
iab
ilit
y
Condition
Percent Cell Viability under various conditions 48 hours post treatment
73
6. CONCLUSIONS
Macrophages could be polarized to different phenotypic states depending on the type of
stimuli used. J77A.1 macrophages were polarized to the M1 phenotype by treatment with
100ng/ml LPS for 6 hours and to M2 state using 100ng/ml IL-4 for 16 hours thus demonstrating
their plastic nature. Multiple emulsions can be reproducibly formulated using safflower oil, Span
80 and Pluronic-127. Multiple emulsion formulated to encapsulate siRNA demonstrated 99.7%
encapsulation efficiency with the siRNA maintaining its stability and integrity after
homogenization based formulation process. The size of oil droplets of the siRNA encapsulating
ME was about 0.8 microns which was close to the size of most natural micro-organisms and thus
easy for recognition and phagocytosis by macrophages. The zeta potential for these emulsions
were between -46 to -47 mV. The encapsulation study done using dextran conjugated tetramethyl
rhodamine confirmed that the emulsion system can encapsulate water soluble payload in the
internal phase without any evidence of leakage into the external phase. Gene silencing studies
carried out using CSF-1R siRNA showed that the siRNA was not able to downregulate the
expression of CSF-1R even at a concentration of 100nM. Cell Uptake studies carried out using
labelled siRNA encapsulated emulsions clearly indicated that he siRNA was taken up by cells.
Thus a possible explanation for lack of activity might be the degradation of siRNA in the
endomsomal-lysosomal compartment.
Next, microRNA 155 encoding plasmid and a null plasmid were encapsulated in the
internal phase of the multiple emulsion system and the size of the resulting emulsions were 0.9
microns which again was close to the size of natural pathogens. The encapsulation efficiency for
miR-155 encoding plasmid and null plasmids were determined to be 60 and 65% respectively. The
absence of plasmid DNA in the external phase of the emulsion indicated that about 35-40%
74
plasmid DNA was lost during the process of formulation possibly due to degradation during the
high shear homogenization process. The DNAse protection assay and gel electrophoresis of
extracted plasmid proved that emulsion system protected it from degradation by DNAse activity
and stability was maintained after encapsulation. Cellular uptake studies using labelled plasmid
showed that a time dependent uptake was seen staring within the first hour after incubation and
gradually increasing up to 6 hours. Quantitation of intracellular miR-155 levels showed that a 3
fold increase 72 hours post treatment. Macrophage repolarization studies showed that the miR-155
was capable of elevating the intracellular level of pro-inflammatory markers like IL-1β (12 fold)
and iNOS (6 fold) 48 hours post IL-4 stimulation indicating the repolarization of macrophages to
a M1 phenotypic state after earlier polarization to M2 phenotype after IL-4 treatment. The
emulsion system was well tolerated by the macrophage as confirmed by the cytotoxicity analysis.
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