PERFLUOROCARBON UTILIZATION FOR OXYGEN DELIVER TO

CANCEROUS CELLS

An Honors Thesis

Presented by

Lawson Ethan Spence

Completion Date:

December 2022

Approved By:

_____________________________________________

Richard Schur, Honors Director

_____________________________________________

Rachael Day, Professor of Biology & Chemistry

_____________________________________________

Drury University, 2022

© 2022, Lawson Ethan Spence

Abstract:
Cancerous cells are often hypoxic and inflamed with these pathways linked at the
molecular level. Restoring oxygen to these areas offers a promising method of influencing
inflammation and enhancing therapeutic methods for cancer treatment. Perfluorocarbon (PFC)
nanoemulsions can be utilized to deliver oxygen to areas of hypoxia due to the increase in
dissolved oxygen concentrations of PFCs compared to water or traditional oil nanomaterials. The
nanoemulsions can be loaded with cargo and delivered throughout the body which gives PFC
materials the possibility of being used for a variety of clinical therapies and offers potential
alternative methods of delivering oxygen to a localized area. This study focuses on inducing
inflammation in cells using lipopolysaccharide (LPS) and monitoring the cellular response by
measuring inflammatory cytokine levels before and after return to normoxia following a PFC
nanoemulsion treatment. Future work will use the methods developed here to treat the cells’
inflammatory state with PFC nanoemulsions to make current treatment for solid tumors more
successful.

Keywords: inflammation, perfluorocarbon, lipopolysaccharide

 2

PERFLUOROCARBON UTILIZATION FOR OXYGEN DELIVERY TO

CANCEROUS CELLS

Introduction
Perfluorocarbons & Cell Hypoxia

The fight against cancer is ongoing, and many treatments in the clinic use
chemotherapeutics to promote cancer cell death.1 These treatments fail to treat the underlying
hypoxia and inflammation found in solid tumors,2 which can often result in solid tumors that are
or become resistant to traditional chemotherapeutics. The hypoxic state of tumors has been
linked to inflammation, hence a tumor that lacks oxygen is often also inflamed. By delivering
oxygen to a tumor and preventing hypoxia, it is expected that inflammation will be reduced
resulting in tumors that become more susceptible to traditional chemotherapeutics that often rely
on the presence of oxygen. To induce an inflammatory response cells can be treated with varying
concentrations of lipopolysaccharide at many time points. The inflammation of the cells can also
be quantitatively analyzed by measuring the levels of transcription factors IL-6, TNFα and
NF-κB. Perfluorocarbons, molecules in which all C-H bonds are replaced with C-F bonds
(Figure 1A) can then be utilized to deliver oxygen to these hypoxic/inflamed cells returning them
to normoxia. Perfluorocarbons have desirable characteristics as synthetic oxygen carriers due to
the compact size of PFC particles that exhibit 20 times the solubility of gasses compared to water
and bind 40-50 times more oxygen than hemoglobin.3

Stabilizing Perfluorocarbon Nanoemulsions:

PFC nanoemulsions are droplets of fluorous solvent stabilized by surfactants dispersed in

2
water. Stabilizing these nanoemulsions makes them into long-lasting structures, rather than
assembling and disassembling transiently. Stabilization of these emulsions offers a material that
can be loaded with cargo and delivered to a specific area. The size, stability, and surface
chemistry of PFC nanoemulsions is controlled by the surfactant that is used to stabilize the
nanoemulsions. One study from Mayes, et. al. uses an acrylate polymer with PFC and
poly(oxyethylene) ester group to stabilize an emulsion of functional monomer, cross-linker, print
molecule, initiator, and solvent in a more atypical PFC material, perfluoro(methylcyclohexane).
UV irradiation of this combination of polymer and perfluorocarbon resulted in polymer
beads/emulsions that could be controlled between about 5-50 μm by varying the amount of
stabilizing polymer.9 These polymers gave the beaded packings low back pressure and rapid
diffusion and good separation even at high flow rates. Figure 1 shows the formation of
nanoemulsions with the use of sonication. A mixture of PBS and 20mL of perfluorooctyl
 3

bromide are sonicated for about two minutes to form the emulsions that are found to average
200µm in diameter (Figure 1B/C).

Figure 1. A) Comparison of hydrocarbon and perfluorocarbon structures. B) Stabilization of PFC in nanoemulsions

using sonication. C) Pluronic F68 (PF68), an amphiphilic polymer used to stabilize perfluorooctyl bromide (PFOB).

Perfluorocarbons' Utility in Clinical Treatments

Perfluorocarbons have proved useful in many areas of clinical treatment. The most
well-known characteristic of these materials is their oxygen-carrying capacity, which has allowed
perfluorocarbons to be used as an alternative blood supply for delivering oxygen throughout the
body.17 Perfluorocarbons have also been used to enhance the performance of hepatic cultures,5 in
hepatic cells it is often difficult to deliver oxygen within their three-dimensional configurations.
A PFC oxygen carrier was added to significantly increase the oxygen capacity in a medium.5
This study clarifies the benefits of using PFCs to enhance the functional performance of
three-dimensional liver systems by analyzing oxygen perfusion in a customized tissue assembly
system. The results demonstrated that the addition of PFCs significantly increased the oxygen
capacity of regular medium and that net cytochrome P450, which is responsible for the
metabolism of drugs and other xenobiotics, showed activity levels that were considerably
increased underflow in PFC-treated systems as compared to controls.5 This study allows for an
example of utilizing these nanoemulsions to a more localized source, rather than producing
systemic oxygenation.
Perfluorocarbon liquids have been used to facilitate surgery in a multitude of conditions.
More specifically, in ophthalmology cases including proliferative vitreoretinopathy, retinal tears,
drainage of suprachoroidal hemorrhages, diabetic traction,6, and retinal detachment.7 PFC liquids
interact with light in a similar manner to water due to their comparable refractive indexes. This
 4

allows the use of a normal contact lens for vitreous surgery while the low viscosity facilitates
tissue manipulation, injection, and removal.6 Peyman and Sullivan found that PFC liquids are not
tolerated in the anterior chamber of the eye, and often caused corneal edema within 2-3 days at
the site of contact. Although PFCs are considered biocompatible, this is an example of where
PFCs have been shown to have unintended side effects. Understanding the potential
consequences of PFC administration is vital before clinical trials or the use of any therapeutic
methodologies. Finding these side effects allows us to alter the methods of PFC administration to
create a more reliable and specific form of treating disease with PFCs. Another ophthalmological
case where perfluorocarbon gasses were utilized in the treatment of retinal detachment.7
PFCs have also been formulated into nanomaterials for use in Photodynamic therapy
8
(PDT). This therapy uses oxygen, light, and a photosensitizer (Figure 2A) to create cytotoxic
singlet oxygen. In tumors, pre-existing hypoxia and oxygen consumption during PDT can
hamper photodynamic efficacy. To overcome this problem, this study loads a photosensitizer into
perfluorocarbon nanodroplets. This significantly enhances the effect of the photosensitizer. 15
They found that PFC nanoemulsions greatly increase the efficacy of photosensitizers in treating
cancer (Figure 2B).

Figure 2. A) Common photosensitizers used for photodynamic therapy (PDT). B)Tumor inhibition from PDT utilization with
simultaneous PFC oxygen delivery utilization

Perfluorocarbons (PFCs) for Systemic Oxygen Delivery

Many different blood substitutes can be used as an alternative method of delivering

oxygen throughout the body when endogenous hemoglobin is insufficient. Once oxygen is
delivered to specific tissues that are relatively hypoxic the carrier vehicle releases oxygen into
the lower concentration through simple diffusion. Blood substitutes can also be utilized to
disperse gasses other than oxygen systemically.11 The transport of nitrogen and other gasses
 5

throughout the body is also an important ability of any load-carrying blood substitute like PFCs
and should be analyzed in comparison when describing a compound's oxygen-carrying capacity.
PFCs not only provide effective gas exchange but also acid/base balance and improve
lung function. PFC ventilation has been suggested as an especially effective therapy for severe
respiratory distress in human infants.16 The purpose of this work was to determine the optimal
volumes of PFC to be delivered, the frequency of the ventilatory cycle, the oxygen flow rate, and
the best circuit set up for neonatal application. The results showed that total liquid ventilation is a
valid alternative to gas ventilation. Liquid ventilation was found especially effective in cases
involving infants with insufficient or no production of surfactant in the respiratory system.
Oxygen delivery has been established in animal models and through Phase II and III
human clinical trials.12 Fluosol-DA was approved by the FDA specifically for the perfusion of
ischemic tissues in percutaneous coronary intervention in the 1980s and 1990s. Multiple
fluorocarbons have been investigated for use as oxygen carriers, including lipophilic and polar
PFC structures. Fluorocarbons containing lipophilic elements are excreted more rapidly than
what would be predicted of their molecular weight. PFCs used for oxygen delivery should be
extremely hydrophobic to prevent undesired interactions with biological media. It has been
found that the body excretes perfluorocarbons via exhalation after hydrolysis (Figure 3).
Perfluoro-based oxygen carriers take advantage of high-solubility respiratory gasses
when they are formed into nanoemulsions. There are currently no perfluorocarbon-based oxygen
carriers approved for clinical uses. Many of these substances have been attempted for approval
but failed due to secondary effects of the surfactants employed, like Fluosol DA.19 Others, such
as Oxygent, were found to have adverse cerebrovascular effects on cardiopulmonary bypass. One
study from Castro and Barceno reviewed the most common perfluorocarbons used for oxygen
delivery and found specific causes for failure to be approved for clinical use. Some PFCOCs
have failed due to secondary effects of the surfactants employed, like Fluosol DA, whereas
others to adverse cerebrovascular effects on cardiopulmonary bypasses, such as Oxygent.19
Further research is needed to develop strategies to counteract these compounds’ shortcomings.
 6

Figure 3. Intravenous PFC Nanoemulsions are eliminated from the body through exhalation

Key issues related to the selection of an appropriate, readily excretable PFC and the
engineering of a stable injectable PFC emulsion have also been analyzed.13 Oxygent (PFC
emulsion made primarily of F-octyl bromide) is efficacious in avoiding and reducing red cell
transfusion during surgery. This study found no possible interactions between Oxygent and fluids
present during cardiopulmonary bypass surgery and no effect from the emulsions on hemostasis,
hemolysis, and blood rheology.13
PFCs can be specifically interchanged as a blood substitute in order to reduce allogeneic
blood transfusions or to improve tissue oxygenation.17 One study also states that additional uses
of PFC emulsions can include treatments of diseases with compromised tissue oxygenation
(cerebral/myocardial ischemia, air embolism, emergency/trauma surgery) as long as no
allogeneic blood is available.17 The study also states the use of PFC emulsions can augment
tumor oxygenation to render these tumor cells more sensitive to radiation and chemotherapy. The
study also finds that the relatively short half-life of PFC emulsions (< 24 h) will not compromise
their success in reducing the requirement for allogeneic blood transfusions. 17
Research from Dr. Krafft at the University of Strasbourg looks at methods of reducing
hypoxia in tumors with perfluorocarbon-based oxygen carriers (Figure 4). Hypoxia of cancerous
cells is a major problem when attempting various cancer treatments.18 Most cancer treatments
require oxygen for the degradation of the tumor with reactive oxygen species. Liquid
perfluorocarbons are solvents that allow to the restoration of normal oxygen levels to these
hypoxic areas. Local oxygen delivery with the nanoemulsions occurs facilitated by ultrasound.
The nanoemulsions can also be loaded with alternative cargo that could aid in the cancer
treatment process.18 These emulsions can help deliver small molecules such as
 7

chemotherapeutics and photosensitizers. This study concludes by finding that PFC-based carriers
may provide new strategies for improving immune responses in cancer patients.

Figure 4. Oxygen-loaded nanoemulsions can be used to deliver oxygen to a hypoxic environment.

Diminishing tumors’ hypoxic state through the administration of perfluorocarbon oxygen

carriers can be utilized in addition to other therapeutic remedies to treat a cancer diagnosis. A
study from Krafft, M.P. shows that PFC-based oxygen carriers increase the lifetime of oxygen
and can enhance photodynamic therapy, along with radiotherapy and chemotherapy.21 The use of
PFC nanocarriers can promote T-cell infiltration, which is an important aspect of the body’s
immune response toward cancerous cells. Hypoxia is a major problem for most cancer treatments
that require oxygen to generate reactive oxygen species that destroy tumors.21 The local oxygen
delivery is facilitated by the use of ultrasound.21 PFC nanocarriers can also be loaded with
fluorescent dyes, photosensitizers, or loaded with chemotherapeutics. Lastly, the study from Dr.
Krafft finds that perfluorocarbon payload carriers could also provide new ways to promote T-cell
localization towards tumors to return a greater and more specific immune response.
Cell death, or apoptosis, can occur from an overactive immune response to foreign
pathogens. Immunogenic cell death causes the release of tumor-associated antigens and triggers
other immune cells to exhibit an anti-tumor immune response. Immunogenic cell death-inducing
medicines exhibit a strong potential for enhancing chemotherapy in clinical applications.1 This
review shows nanoemulsions' characteristics for mediating an immune reaction during any
chemotherapeutic treatment in response to cancer diagnosis.
 8

Potential PFC Utilization Side Effects

Phagocytosis of PFC emulsion particles leads to normal immunological responses via an

innate host-defense mechanism. Stimulation of macrophages releases intracellular products
(metabolites of the arachidonic acid cascade and cytokines).20 These products are responsible for
more of the biological effects that come with PFC emulsion use (cutaneous flushing, fever at
lower doses, and macrophage hypertrophy and recruitment at higher doses). These effects do not
result in any permanent tissue alteration, even with prolonged exposure at relatively high doses.
One study analyzing side effects from PFC therapeutics finds that the emulsions may elicit minor
untoward effects, but that these effects are reversible and, at clinically relevant doses, do not
pose a toxicologic risk.20

Lipopolysaccharide-Induced Inflammation and Alternative Methods

Lipopolysaccharides

Lipopolysaccharides (LPS) are large molecules that consist of a lipid with

a polysaccharide which is a toxin produced by bacteria. 31 LPS is an
immune system activator LPS acts as an endotoxin and binds the
CD14/TLR4/MD2 receptor complex in many cell types, but especially in
monocytes, dendritic cells, macrophages, and B cells, which promotes the
secretion of pro-inflammatory cytokines, nitric oxide, and eicosanoids. 32

Figure 6. Lipopolysaccharide (LPS)

Figure 7. Brefeldin A (BFA)

Brefeldin A

Brefeldin A (BFA) is a lactone antiviral produced by the fungus Penicillium

Brefeldianum.33 Brefeldin A inhibits protein transport from the endoplasmic reticulum to the
Golgi complex by indirectly preventing the association of a COP-I coat through inhibition of
guanine nucleotide exchange on a Guanine Nucleotide Exchange Factor (GEF) called GBF-1,
which is a part of the Arf family of GEFs.34 Rat pneumocytes produce macrophage inflammatory
protein-2 (MIP-2) and other inflammatory proteins such as interleukin-6 in response to
 9

lipopolysaccharide (LPS) stimulation. Elevated MIP-2 levels were linked to increasing

microtubule depolymerization, which is necessary to maintain cellular structure and normal
morphology. One study found that brefeldin A, by blocking anterograde transport from the
endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture
medium and increased its storage in cells. This study was used to determine necessary BFA
concentrations to reduce LPS-induced microtubule depolymerization.35

Figure 8. N. Isowa - Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes (35)

Varying levels of LPS can induce different levels of inflammation. Liang and Baudouin
evaluated and compared the effects of lipopolysaccharide in which three rabbit corneal injury
models were utilized for comparison and were tested using corneal incision, corneal epithelium
scraping, and corneal suture. Ten rabbits were used in each model and divided into subgroups,
with or without LPS exposure, where saline was administered for a control group. The study
found that LPS induced earlier and higher levels of inflammation in eyes that were subjected to
scraping and suturing compared to saline, showing that the LPS induced a larger amount of
inflammation.22 The results indicated that LPS is a potent proinflammatory stimulus and its
exposure has major effects on inflammation, angiogenesis, and apoptosis.22
Multiple quantitative measurements can be utilized to show varying levels of intracellular
inflammation. Analyzing inflammation as a biological response of the immune system is an
important characteristic to quantify any inflammatory response. Inflammation can be triggered
by pathogens, cellular damage, and toxic compounds. These can induce acute or chronic
inflammatory responses throughout the body. Inflammatory signaling pathways become
activated during this time, most commonly the NF-κB, MAPK, and JAK-STAT pathways. One
study reviews altering inflammatory responses in various organs, looking at organ-specific
inflammatory responses.23 The study also shows the NF-κB pathway which is triggered by TLRs
and inflammatory cytokines, like TNF and IL-1. NF-κB signaling also requires IKK subunits that
regulate the activation of the pathway through IκB phosphorylation.23
 10

Other studies have looked at the inflammatory response in multiple cell types.
Cardiomyocytes can also be induced to become inflamed and analyzed. In this setting, cells were
treated with lipopolysaccharide (LPS) at varying concentrations and times. It was found that LPS
inhibited the conductance of calcium-activated potassium channels and enhanced
sodium/calcium exchange (antiporter).24 The LPS also induced prolonged action potential
duration which suggested electrical dysfunction in the cellular environment. 24

Inflammatory Response in Cells (Transcription Factor Activation) HIF and NF-κB

The hypoxic environment stemming from inflammation plays an important role in many
diseases. Mammals are seen to have developed conserved programs of the transcriptional
response to the hypoxic environment and inflammation.25 Both of these characteristics
commonly occur together, and transcription factors are able to respond to these cellular
characteristics. Current understanding of an immune response shows the crosstalk between the
cellular hypoxic response and inflammatory transcription factors are simultaneously present. One
study specifically analyzes these pathways' presence in rheumatoid arthritis, inflammatory bowel
disease, and colorectal cancer.25 It is also found that individuals with mountain sickness have also
presented with increased levels of inflammatory cytokines, specifically cytokine IL-6.25
An oxygen-rich environment is inherently necessary for aerobic life at the cellular level.
Aerobic life is dependent on molecular oxygen for ATP regeneration through oxidative
phosphorylation.26 This is only possible in a small range of oxygen concentrations. Variations in
the levels of oxygen are toxic and produce reactive oxygen species (ROS).26 Cells have been
found to have developed strategies to respond to changes in this oxygen concentration in which
specific transcription factors become activated and target genes are transcribed. HIF-1
(hypoxia-inducible factor 1), a transcription factor that responds to a cells’ hypoxic environment,
and NF-κB are regulated. There is speculation on the intracellular mechanism for the
determination of this change in oxygen levels in the cellular environment.26 HIF-1 as a response
to a hypoxic environment is especially present in malignant melanoma.
Inflammation was initially seen early in human history and was characterized in Latin as
having features of heat (calor), pain (dolor), redness (rubor), and swelling (tumor).37 Modern
scientific analyses lead to the characterization of inflammation at the molecular level. As
discussed, there are immediate changes in intracellular transcription factor activation, including
HIF-1 and NF-κB. Acute inflammation is seen to trigger an increased liver synthesis of positive
acute-phase proteins (APPs).27 The serum level of these APPs later returns to a base
concentration when the factor that caused it is no longer present. In clinical treatments of an
inflammatory patient, diagnoses are made by analyzing specific markers such as the presence of
these mentioned inflammatory markers. Acute-phase C-reactive protein
(CRP) is a vital protein to include in an assessment of any clinical inflammation as its
determination can be used as an inflammatory biomarker in many diseases.27
 11

The production of HIF-1 and NF-κB mediate the cell's transcription response to a
hypoxic environment. A lack of HIF-1 production has been found to be associated with disorders
such as chronic hypoxia from pulmonary hypertension.28 A study by BelAiba et. al. found that
hypoxia transiently elevates the production of HIF-1 mRNA and was found to have the
maximum production at one hour after the hypoxic event.28 The study found that deletion of the
HIF-1 promoter contained the genetic region responsible for a response to hypoxia. The cells’
hypoxic state induced nuclear translocation of the NF-κB subunits p50 and p65. Inhibition of
NF-κB diminished hypoxic induction of HIF-1, mRNA, and later protein synthesis. The study
concludes that HIF-1 is a transcriptional target of NF-κB which is activated from a PI3 Kinase
pathway under hypoxic conditions.28
Macrophage concentration in hypoxic areas and their reaction to normoxia are also key
factors in understanding the mechanisms of how hypoxia has an influence on immunity. Data
shows how the hypoxic environment often creates variations in macrophages' phenotypes and
their overall plasticity.29 It was found that hypoxia generated a regulated control of macrophages'
phenotypic plasticity, and further research will find new effective ways to manage diseases with
hypoxic disturbances.
The α subunit of HIF-1 is the master activator of the cellular transcriptional response to
hypoxia and propagates the gene expression that is induced during a hypoxic cellular
environment. Overexpression of this protein is linked to a progression of many tumor types
(head/neck, cervical carcinoma, leukemia, and renal cell carcinoma).30 This study from Kuphal
demonstrates that HIF-1 activity is increased in malignant melanoma cells that are also under
normal oxygen concentrations. This effect is because of the constitutive HIF-1 expression, which
is the default pathway for transport and is an unregulated pathway. The inhibition of the NF-κB
pathway lowered the intracellular accumulation of HIF-1 and also suggests that reactive oxygen
species along with NF-κB activity are responsible for this HIF-1 accumulation.30 This study
concluded that the overproduction of HIF-1 in melanoma is mediated by reactive oxygen species
and the NF-κB pathway. 30

Results and Discussion

Here we develop a reliable method to induce cellular inflammation so that it can be

determined if PFC nanoemulsions administration will reduce the inflammatory state while
simultaneously returning cells to normoxia. The cell's inflammatory state is first induced to a
similar level seen in cancer cells to prevent apoptosis.36 The cellular inflammatory state can be
quantified by examining cytokine levels such as IL6, NFκB, and TNFα before and after
treatment. Mouse macrophage cells (RAW264.7) are treated with LPS (0-10 ug/mL) and
Brefeldin A (BFA) (300 ng/mL) for 48 hours (37°C, 5% CO2). BFA inhibits protein transport
from the endoplasmic reticulum to the Golgi complex. In the treatment, BFA is used to reduce
LPS-induced microtubule depolymerization. This allows for the inflamed cells not to undergo
 12

cell death so that a valid measurement of their inflammatory state is able to be examined (Figure
5).

Figure 5. Inducing Inflammation using LPS + BFA

The levels of inflammatory cytokines were measured via qPCR and were found to have
increasing levels of inflammation starting with a concentration of 0.1µg/mL and even more
inflammation with 10µg/mL (Figure 9). The findings from this qPCR data were further
supported after running an agarose gel electrophoresis to visualize the data qualitatively by
bandwidth (Figure 10).

Figure 9. qPCR fold change relative to β-Actin measurements in cell samples ranging from 0.01µg/mL to 10µg/mL LPS with
Brefeldin A added in all samples at equal concentrations.
 13

Figure 10. Agarose Gel Electrophoresis to analyze qPCR DNA quantities from Figure 8 data. IL-6 was found to have increased
bandwidth as LPS concentration increased.

This gel allowed for the visualization of the marker IL-6, which was increased
corresponding to higher levels of LPS treatment; the increased level of interleukin-6 indicated a
higher level of inflammation expressed in the RAW 264.7 cells. The experiment was repeated to
confirm the inflammatory state in response to LPS exposure. Once it was confirmed that
inflammation was being induced, it was determined that a concentration of 0.1µg/mL was
sufficient to create the level of inflammation necessary to examine cells comparable to a level of
inflammation that would be found in cancerous tumor cells.

Figure 11. qPCR fold change relative to β-Actin measurements in cell samples with 0 LPS added or with 0.1 µg/mL LPS with
and without perfluorocarbon nanoemulsions added.

After determining an ideal LPS concentration of 0.1µg/mL, perfluorocarbon

nanoemulsions efficacy was tested through administration to inflamed cells to test for a reversal
to their basal state of inflammation. The qPCR data indicated that the emulsions did not reduce
the inflammatory markers to reverse the cells’ inflammatory state to a basal level that would be
expressed without LPS exposure (Figure 8). An agarose gel was performed to further visualize
the DNA levels within the cell at the time of sample collection (Figure 9). The agarose gel was
 14

run with the control sample compared to the 0.1µg/mL LPS treatment to again ensure that
inflammation was being induced. The emulsion sample was not placed into the gel due to a lack
of indication of effectiveness.

Figure 12. Agarose Gel Electrophoresis to analyze qPCR DNA quantities from Figure 8 data. IL-6 was found to have increased
bandwidth as LPS was given even if less sample was loaded into the gel according to the β-Actin well.

To continue to optimize an ideal method of inducing inflammation, various time

treatments were given to the cells with consistent levels of LPS concentrations (0.1µg/mL). Time
exposures ranged from 0 hours as a control group up to a 48-hour exposure. For the three-hour
time points, two samples were created. One 3-hour treatment received a 0.1µg/mL LPS
concentration, while the other sample received a 10µg/mL LPS concentration. A qPCR was
performed to analyze levels of interleukin-6, NF-κB, and TNFα. This qPCR data indicated that a
high level of inflammation was able to be induced in a short duration of just three hours (Figures
13 and 14). It was observed that at 48 hours the cells presented with a lower level of
inflammation due to cell apoptosis from overexposure to LPS. Interestingly, at 24 hours the cells
began to exhibit apoptotic characteristics and a high level of metabolism indicated by an
extremely yellow media color in the sample. The experiment was duplicated two more times to
confirm inflammation was induced in the 3-hour time treatment.
Figure 13. qPCR fold change relative to β-Actin measurements in cell samples with 0.1µg/mL LPS at varying time
points including 3 hours, 24 hours, 48 hours, and an additional 3-hour treatment with a high concentration of LPS to test for the
enhanced inflammatory state in a short duration of time.
 15

Figure 14. qPCR fold change relative to β-Actin measurements in cell samples with 0.1µg/mL LPS at varying time points
including 3 hours, 24 hours, 48 hours, and an additional 3-hour treatment with an ultra-high level of LPS concentration to test for
the enhanced inflammatory state in a short duration of time. The second chart is a repeated experiment without the high-level
LPS concentration for 3 hours.

This qPCR data indicated that a high level of inflammation was able to be induced in a
short duration of just three hours. It was observed that at 48 hours the cells presented with a
lower level of inflammation due to cell apoptosis from overexposure to LPS. Interestingly, at 24
hours the cells began to exhibit apoptotic characteristics and a high level of metabolism indicated
by an extremely yellow media color in the sample. The experiment was duplicated two more
times to confirm inflammation was induced in the 3-hour time treatment.
The experiment was repeated without the 48-hour treatment, due to repeated attempts
confirming that inflammation was more consistent in the 3 and 24-hour treatments (Figure 15).

Figure 15. qPCR fold change relative to β-Actin measurements in cell samples with 0.1µg/mL LPS at varying time points
including 3 hours and 24 hours

A Luciferase assay was performed to test whether the hypoxic state of the HEK293T cells
was able to be induced through the LPS/BFA exposure method that had been developed utilizing
 16

the RAW264.7 cells from the qPCR data. The Luciferase assay indicated that the
hypoxic-dependent luciferase enzyme was inactive when exposed to LPS and BFA. This
indicated that although the inflammatory markers were expressed through this method, another
form of inducing the hypoxic pathway will be required, or rather an alternative method of
inducing both pathways simultaneously.

Figure 16. HRE dependent and standard Luciferase fold change relative to a control group not transfected with Luciferase gene

Materials and Methods

RAW264.7 cells (400,000 cells/well, 6-well plate) are treated with LPS (0-10 ug/mL) for
48h at 37°C. After 48 h treatment with LPS, BFA was added (300 ng/mL, 3 h). Following
treatment, cells were washed, and RNA was purified from each sample using the xx kit. The
RNA was reverse transcribed to cDNA for qPCR analysis of inflammatory cytokine. Afterward,
samples were loaded into an agarose gel for visualization.
The experiment starts by plating equal cells onto a 6-well plate. The cells are cultured
until about 50% confluent and then treated with LPS. The LPS treatments would vary in a
concentration ranging from zero as a control group up to 10 µg/mL. After 48 hours of treatment
with LPS, Brefeldin A would be added at a concentration of 300 ng/mL. After 3 more hours, the
cells were lysed and the RNA was purified from each sample. The RNA samples are then loaded
onto a nanodrop to measure RNA concentrations and then standardized to equal amounts and
prepared for reverse transcription. After reverse transcription, the cDNA samples undergo qPCR
to quantify relative levels of RNA that were present within the cell. Afterward, the samples from
qPCR are loaded into an agarose gel for electrophoresis for a visualization of the relative levels
of the desired markers.
 17

Broader Impacts

To utilize perfluorocarbons on a regular basis, further work needs to be completed. With

hopes to use these as an alternative method to deliver oxygen to cells undergoing any sort of
hypoxic event is a therapeutic strategy that is still in the beginning stages of development.
Maintaining emulsions that have been formed through sonication for storage, and loading them
with their specific cargo, in this case, oxygen is another obstacle preventing their widespread
usage, as seen by the recall of Fluosol-DA as a blood substitute. Then, determining methods of
delivery in clinical use and understanding the pharmacokinetics will prove useful to eliminate
any unintended side effects.
These materials have shown promising characteristics for their usage in other various
clinical applications. These materials’ inherent cargo-carrying capabilities could prove useful in
other medicinal therapeutic treatments besides systemic oxygen delivery. With the continuous
research in this field, it is likely that the development of an efficacious method of PFC utilization
will be created and utilized in the near future.
 18

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