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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus

Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)

Anionic Liposomal
Encapsulation of Oncolytic
Adenovirus
Nicolas Etcheverry (A08662261)
Vanessa Herrera (A09202452)

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
A) Objective, Hypothesis and Aims
Objective
The encapsulation of oncolytic adenoviruses within anionic liposomes with surface
receptors offers an alternative novel therapeutic modality to target cancer types present
in areas of the body that are difficult to treat using conventional methods. Cancer
encompasses a broad group of various diseases involving unregulated cell growth. It is
a medical condition that has high incidence and mortality rates. Patients undergo harsh
treatments that can potentially lead to severe side effects. Conventional cancer
treatments include chemotherapy, radiotherapy, and surgery. These treatments are
often inefficient, expensive, and time-costly. Apart from tangible costs, there is a
significant amount of emotional trauma experienced not only by the patient, but also by
their family. Even mild side effects of conventional treatments, such as hair loss, can
bring the patient intense emotional hardship.
The expected outcome from the encapsulation of the oncolytic adenovirus includes the
potential to lead to an alternative therapeutic approach for cancer treatment. The
efficient transfection and subsequent lysis of the cancer cells by the oncolytic virus can
reduce the tumor size and enhance the probability of successful removal by surgery.
Moreover, if the tumor is small and at an early stage of development, complete
disintegration of the tumor with the virus can be possible. The main limitations of
oncolytic viruses are that they can be neutralized in the blood circulation by antibodies
and they cannot transfect in cancer cells that do not have coxsackievirus and
adenovirus receptor (CAR). Encapsulation of the viruses will eliminate these limitations.
This therapeutic strategy can target many types of cancer and can be used to treat
cancers that have low survival rates using other conventional treatments. Encapsulation
of oncolytic adenoviruses in anionic liposomes with surface receptors can enhance
localized delivery, reduce immune response, and transfect CAR-negative cell lines in
order to effectively reduce tumor size.
Specific Aims
Our research aims to significantly enhance and bring innovation to cancer
treatment. Our potential product brings a novel approach with limited side effects
compared to conventional treatments. The commercial application will be huge, as
cancer is the leading cause of death in the United States. Our treatment would quickly
take a large percentage of market share from less effective conventional treatments.
A. Develop an efficient oncolytic adenovirus encapsulation procedure
A1. Formation of liposomes via lipid film hydration
The lipid film hydration process will be utilized to encapsulate the oncolytic viruses.
A2. Characterization of the Liposome

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
The Dynamic Light Scattering (DLS) technique will be used to characterize the size,
charge, and PDI of the liposomes. Through Scanning Electron Microscopy (SEM), we
are interested in observing the morphology and size distribution of the liposomes.
A3. In vitro viral transfection testing in the presence or absence of serum
The transfection of CAR-positive and CAR-negative cell lines will be tested with naked
adenovirus, encapsulated adenovirus, and empty liposomes (no adenovirus) in both
serum-free and serum conditions.
B. Purification of liposomes
B1. Filtration of large (>300nm) and small (<200nm) liposome nanoparticles
To narrow the range of the size of liposome nanoparticles, large nanoparticles can be
separated by employing a pressure filtration syringe while size exclusion
chromatography is applied to retain the unwanted small liposomes.
B2. Immunoprecipitation of non-encapsulated viruses
To purify the sample of any free-floating adenoviruses in the solution, an
immunoprecipitation technique will be employed.
C. In vivo mouse model testing
C1. Intravenous Injection of encapsulated nanoparticles with controls
The anionic liposomes with adenovirus will be injected intravenously into mice models
with tumors to test the tumor attacking efficiency of the adenovirus delivering
constructs.
C2.Biodistribution of virus using histological Analysis
The distribution of the injected viruses in multiple organs including the liver, lungs,
intestines, etc. will be detected using immunohistochemistry.
C3. Macroscopic analysis of tumor size
At determined time points, the mice will be sacrificed and tumors will be removed to
measure their size reduction.
B) Background and Significance
Despite advances in chemotherapy, radiation therapy, and surgical procedures,
mortality rates for late-stage cancer remain
high. Furthermore, severe side effects from
such therapeutic strategies deteriorate
patients health and life [1]. Thus, there is
an increased need for a novel therapeutic
approach that allows for the successful
reduction of both primary tumor and
metastasis. One such novel strategy is
oncolytic viral therapy. Oncolytic viruses
are viruses that selectively replicate in
cancer cells, but spare normal cells [2].
Figure 1. Schematic diagram of the cancer-selective killing of
oncolytic adenovirus. Eunah Kang, BMB Reports 2010.
Oncolytic viruses are an attractive cancer
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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
treatment alternative because they can only lyse cells with deficiencies in the p53 tumor
suppressor molecule, a mutation commonly seen in cancer cells (Figure 1).
Additionally, since viruses have replication capabilities, a small dosage can lead to an
increase therapeutic effect [2].
Oncolytic viral therapy has been a working hypothesis for a long time. In the past two
decades, several advances have been made in the field. More recently, oncolytic
adenovirus H101 received marketing approval for cancer treatment in China [3].
Although oncolytic viral therapy offers a promising alternative for cancer treatment,
there are still several hurdles that need to be overcome. Limitations such as
neutralization by antibodies mediated by the immune system, rapid clearance from the
blood circulation by the reticuloendothelial system in the liver, and limited transduction
efficacy in cell types that lack the expression of viral surface receptors reduce the
therapeutic potency of oncolytic viruses [1]. With the aim to overcome an immune
response and to enhance its potential
use to treat primary and metastatic
tumors, a method for liposomal
encapsulation of oncolytic
adenoviruses is proposed. The
encapsulation procedure will consist of
an anionic non-toxic lipid, cholesterol,
Figure 2. Schematic of adenovirus encapsulation.REFERENCE
polyethylene glycol (PEG), and a
targeting ligand, as shown in figure 2. An anionic lipid was chosen as the virus carrier
due to their limited nonspecificity capabilities. The resting membrane potential of most
human cells is in the order of negative millivolts [4]. If a cationic lipid was used instead
of the proposed anionic lipid, the liposomes would be attracted to any negatively
charged cell in their vicinity. This nonspecificity could lead to higher levels of toxicity in
the patient. The use of cholesterol in liposomes has been reported to increase liposome
stability whereas PEG was chosen due to its ability to be activated and linked to target
ligands that can prolong the circulation time, improve liposome biocompatibility, and
reduce nonspecificity [5, 6]. The targeting ligand will increase liposome delivery to the
cancer cells, and thus, increase the concentration of oncolytic adenovirus in the desired
site. Furthermore, the adenovirus successful cell infection would not be dependent in
the coxsackie virus and adenovirus (CAR) receptor being present on the cells surface
because their entry into the cell would be mediated by endocytosis of the liposome.
Previous Research
Previous liposomal encapsulation strategies have been investigated by the center of
cancer and gene therapy in Houston [7]. They successfully encapsulated the oncolytic
adenovirus dl1520 in a cationic liposome composed of DOTAP (cationic lipid) and
cholesterol. The encapsulated viruses were able to infect both CAR negative and CAR
positive cancer cell lines, whereas the naked adenovirus was not able to infect the CAR
negative cells. Also, the encapsulated viruses were not neutralized by antibodies and
were able to lyse the cancer cells in the same amount of time than in the absence of
antibodies. The naked adenovirus in the presence of the neutralizing antibodies took
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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
longer to lyse the cancer cells. Even though their results were successful, a drawback
for this liposome formulation is that the viral biodistribution in vivo was concentrated in
the lungs of mice which were injected intravenously with the liposomes. This could be
due to the attraction between the positively charged liposome and the negatively
charged lung cells (resting membrane potential of lung cells ~60mV). Moreover, the lack
of a targeting ligand in the formulation decreased the viral concentration in the cancer
cells, diminishing the lytic potency of the oncolytic virus.
Similar work involving the delivery of an adenovirus using anionic liposomes with
conjugated folate particles to target cancerous epithelial cells lining the lungs was
conducted by the Key Laboratory of Drug Targeting and Drug Delivery Systems at the
West China School of Pharmacy [8]. The work highlighted the use of an anionic
liposome as an encapsulation method for the adenovirus instead of the positively
charged cationic liposome because the upper-airway epithelial cells, covered by a
negatively charged mucus layer, would often trap and neutralize the cationic liposomeadenovirus complexes. The use of the liposomes removed the need for CAR+
receptors in order for cells to uptake the adenovirus, and the folate particles conjugated
on the liposomes allowed for specific cancer cell targeting within the lungs.
Overexpression of folate receptors on human tumors encouraged this specific targeting.
With respect to weaknesses, the results achieved were a result of in vitro cellular
transfection based assays, and further steps involving in vivo mouse models would be
required to further support the results: ligand conjugated anionic liposomes with
adenovirus enhance transfection into tumor cell lines in lung tissue. This paper strongly
relates to our proposed effort because it employs a very similar strategy to target and
attack cancer cells, but slight modifications to the liposome composition and ligands
could enhance tumor size decreases elsewhere in the body.
Miller et al. examined the effect of liposome surface charge on liposomal binding and
endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a
murine derived mononuclear macrophage cell line (J774) [9]. Using large unilamellar
liposomes with or without the addition of positively charged lipids or negatively charged
lipids, where anionic or neutral PEG was added in some experiments, it was found that
HeLa cells were found to endocytose positively charged liposomes to a greater extent
than either neutral or negatively charged liposomes. However, this preference was not
lipid-specific since inclusion of a cationic cyanine dye imparted a positive charge in
place of the positively charged lipid and resulted in a similar extent of endocytosis. On
the other hand, the extent of liposome interaction with J774 cells was greater for both
cationic and anionic liposomes than for neutral liposomes. The greater uptake of
positively charged liposomes by HeLa cells was also observed with sterically stabilized
liposomes (PEG liposomes). It was also found that positively charge PEG liposomes
had a much greater extent of endocytosis compared to negatively charged PEG
liposomes. Replacement of the cationic dye with a neutral dye is crucial to future studies
as the influence of the dye being positively charged is unclear in the results. This
research displays data that contradicts our idea, stating that cationic liposomes lead to
more endocytosis. However, the report does not take into consideration in vivo effects

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
such as the fact that cationic liposomes will interact with non-targeted cells. The use of
PEG will be used in our proposed idea.
Aims and Implications
The specific aims of this research project involve finding an
efficient and stable lipid concentration to use within the
liposome, creating an encapsulating anionic liposome
using standard protocols, and inserting the oncolytic virus
within the liposome. Further research would need to be
conducted in order to examine the over expression of
certain protein receptors on the tumor cells of interest, and
the conjugation of ligands that can bind to the receptors on
the cancer cells would need to be conjugated to the
nanoparticles. All of the functional, chemical, and physical
Figure 3. Folate conjugation to anionic
properties of the liposome/adenovirus complex would need
liposome.REFERENCE
to be understood before moving on to in vitro cellular
transfection testing. When considering the long term plans of the study, the ideal
situation would involve successful in vivo mouse model testing where tumor size
significantly shrinks after systemic delivery of the nanoparticles at the tail vein. If these
tests bode well, successful clinical trials could lead to the ultimate long term objective of
using these encapsulated oncolytic viruses to significantly reduce the size of large
tumors to make surgical removal much easier.
Accomplishing the aims will advance clinical practice by providing a systemic way to
deliver cancer attacking oncolytic virus to all parts of the body by a single injection. In
comparison to invasive treatments such as radiation therapy and chemotherapy, tissue
damage and cell destruction is insignificant. Powerful side effects that accompany
radiation therapy and chemotherapy such as hair loss and gastrointestinal distress can
be avoided [2]. Additionally, this treatment could be a pre-cursor to surgical removal for
first reducing the size of the tumor. This opens up possibilities of avoiding and
increasing the ease of surgical procedures. Understanding which lipids can increase the
effectiveness of the transport liposome in delivery of adenovirus is important for the
growth of scientific knowledge as liposomes as a method of transport in the body have
many applications in treating other diseases.
Our proposed research will show its significance through its improvements in the field of
virus delivery to cancer cells. This project employs the use of an oncolytic virus instead
of drugs, and the virus use is more effective because it can replicate within tumor cells
as it spreads from cancer cell to cancer cell. This prevents the need for high doses of
drugs, and the same effective tumor damage can be accomplished with small doses of
virus. The virus does not attack cancer cells due to its genetic manipulation to focus on
P53 deficient cells. The liposomal encapsulation of the oncolytic virus improves on the
traditional deployment of the naked virus because it allows for systemic intravenous
delivery, prevents an immune response, and allows for passage into CAR negative cells
[4]. The use of anionic liposomes prevents the cytotoxic side effects that often occur
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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
when cationic liposomes bind nonspecifically to cells at a negative resting potential [5].
Most importantly, the use of the conjugated ligand on the liposomal will allow for specific
and direct cell targeting of cancer cells.
Specific scientific questions that have not yet been answered in the field of liposomal
adenovirus delivery to cancer cells include how to purify the sample from nonencapsulated adenovirus prior to treatment and how to determine the appropriate
targeting ligand in the encapsulation procedure. To address the issue of sample
purification, a technique that is able to successfully purify the sample from any nonencapsulated adenovirus has to be developed. Scientific literature typically does not
discuss purification strategies. Purifying the sample before treatment is important since
the non-encapsulated adenoviruses could elicit an immune response that could
complicate the delivery of the liposomes into the target site, thus, reducing the oncolytic
efficiency of the encapsulated viruses [3]. With regards to the field of targeted liposomal
virus delivery to cancer cells, scientists have not yet fully addressed all the possible
ligand-to-liposome combinations that can be effective for cancer cell targeting. While
scientific research has been able to identify which receptors and proteins are over
expressed on the exterior and interior of many different cancer cell lines, there are still a
lot of questions to be answered involving which ligands to these receptors would be
most effective for targeting of the cancer cells without structurally changing the
properties of the liposomal complex. Conjugating the ligands to the nanoparticles can
drastically change the properties of the liposome, and the addition of PEG and other
environmental conditions can have similar effects as well [6]. The work in this project
will definitely address a small portion of this area of scientific knowledge by determining
an ideal combination of PEG, anionic liposomal concentration, unique ligand, and other
possible chemical factors that will result in an effective nanoparticle for our specific
cancer cell targeting purposes.
The commercial opportunities and societal benefits of this project are large. Our idea
would increase the number of treated patients, providing a solution to many cancer
patients who are not receiving positive results in conventional treatments. Many patients
are reluctant to go through invasive treatments such as chemotherapy and opt to go on
without treatment due to the painful side effects of current treatments. Due to the simple
and painless nature of our proposed idea, this population would be easily tapped into.
Many early stage cancer patients would also have the opportunity of exploring new
treatments such as ours. If commercialized, our idea would obtain significant market
share of the quickly growing oncology market which was valued to be $47.7 billion in
2010 [10].
Conclusion
In conclusion, we propose to develop an oncolytic adenovirus encapsulation procedure
that would be able to protect the oncolytic viruses from neutralization by antibodies,
increase their circulation life time in the blood stream, and to infect both CAR positive
and negative cells. The anionic liposomes will decrease the nonspecificity distribution of

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
the liposomes commonly seen in cationic liposomes, and furthermore, preferentially
target a specific type of cancer cell by incorporating targeting ligands in the formulation.
C) Research Design and Methods
Develop an efficient oncolytic adenovirus encapsulation procedure
The lipid film hydration process will be utilized to encapsulate the oncolytic viruses. The
goals of the encapsulation of the adenovirus within the ligand containing anionic
liposome are to promote efficient delivery of the virus to the tumor sites and to prevent
virus neutralization from an immune response in vivo. Potential limitations of using the
lipid film hydration method to form liposomes are that the virus encapsulated per
liposome cannot be numerically determined and that the encapsulation efficiency (EE%)
has been reported to be relatively low [2]. Liposome formation through ether injection
method is an alternative technique that could be used in case the lipid film hydration is
not effective. Any procedure that involves handling live virus should be done in a tissue
culture hood to prevent exposure of the virus to personnel. Organic solvents used in
the liposome formation procedure can be carcinogenic, and thus, considered
hazardous. The mixing step should be done in a chemical hood to prevent exposure
and inhalation of the solvent by the personnel.
The lipid film hydration method starts by dissolving the chosen molar ratios of anionic
lipid, cholesterol, and ligand-PEG solutions in chloroform. The mixing flasks will be
placed in a vacuum overnight to allow the chloroform to evaporate. A dry lipid film in the
bottom of the flask will be seen when the chloroform is completely evaporated.
Subsequent hydration of the dry lipid film with the oncolytic adenoviruses diluted in PBS
will allow for the amphiphilic nature of the lipid to preferentially form liposomes. Viruses
will be encapsulated in the liposomes by being present in the hydration step [1]. To get
a more homogeneous liposomal solution, the sample will be sonicated for 10 minutes
and stabilized in the 4C fridge for 3 hours prior to being used in in vitro or in vivo
studies. The encapsulation efficiency is calculated by EE% = Wt/Wi x 100%; where Wt
is the total amount of virus left in the suspension after encapsulation and Wi is the total
amount of virus added initially [3]. Ether injection method consists of dissolving the lipids
in diethyl ether or ether/methanol mixture. Afterwards, the solution will be injected slowly
to the aqueous solution of adenovirus diluted in PBS under reduced pressure. The
subsequent removal of ether under vacuum leads to the formation of liposomes [2].
To determine if the liposome has the size, charge, and morphology required to have
successful liposomal delivery to cells, characterization of the liposomes prior
experimentation will be assessed. A quantitative analysis of liposomes size and charge
distribution will be determined by the dynamic light scattering (DLS) technique.
Disadvantages of using DLS instrument are that measurements are time consuming, it
can only measure clear (not colored) samples, and it is sensitive to mechanical
disturbances [4]. Scanning Electron Microscopy (SEM) will be employed to observe the
morphology and topography of the liposome suspension. A limitation of using
microscopy is that the images are being subjectively interpreted by the personnel

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
analyzing them. Instead, Anatomic Force Microscopy (AFM) can be used to have a
more qualitative measurement of the liposomal surface topography.
The samples consist of the liposomes formed in the lipid film hydration procedure. DLS
measurements are very sensitive to any particulates like dust, aggregates, and free
bubbles, thus, samples must be prepared carefully. In order to measure charge and size
distribution, a disposable folded capillary cell/cuvette is going to be used. Using a
syringe, the DLS cuvettes will be rinsed two times with ethanol and one time with Milli-Q
water and will be let to dry completely. The appropriate volume of the sample will be
added slowly with the syringe into the DLS cuvette. The surfaces will be wiped off
before placing the cuvette in the DLS instrument. Readings of the samples size and
charge distribution will be taken separately [5]. Liposome samples for the SEM images
will be prepared by dehydrating the sample through ascending series of ethanol ending
in 100% ethanol. Next, the samples will be dried without introducing surface tension
artifacts. After drying, the samples will be mounted on an aluminum stub using silver
paint and will be coated with a very thin film of gold and palladium [6].
To assess if the anionic liposomes are able to transfect cancer cells through a different
entry pathway (not dependent in CAR surface receptor), the particles will be tested in
both CAR-positive and CAR-negative cell lines. We intend to demonstrate that both the
naked and the encapsulated adenoviruses will be able to transfect the CAR-positive
cells, but only the encapsulated adenoviruses will transfect the CAR-negative cells.
Furthermore, through the use of serum containing antibodies for neutralization of the
adenovirus, we will compare the transfection ability of both encapsulated vs. nonencapsulated samples in CAR-positive cell lines. We expect only the encapsulated
adenovirus to transfect the cells since the non-encapsulated viruses will be neutralized
by the serum antibodies. For the studies, green fluorescence adenovirus (GFP-Ad5)
will be used instead of the oncolytic virus to quantify the transfection efficacy of the
liposomes via fluorescence expression. Whenever GFP-Ad5 is replicated inside of the
cells, the cell will express green fluorescence which can be quantified via TECAN
instrument and visualized via fluorescence microscopy. A greater fluorescence
expression means that a better transfection of the cells occurred by the virus. Difficulties
encountered by using TECANs quantification method to assess transfection efficacy is
that background fluorescence should be subtracted from the sample transfection
fluorescence.
The transfection of CAR-positive (eg. HeLa cells) and CAR-negative (eg. CHO cells)
cell lines will be tested with naked adenovirus, encapsulated adenovirus, and empty
liposomes (no adenovirus). This study will be performed by plating HeLa and CHO cell
lines in two separate 96-well plates. After the cancer cells are grown in the plate for 24
hours, the respective samples will be added in triplicates in both plates. Cells will be
incubated for 36 hours before fluorescence quantification. To test for the neutralization
of the viruses exposed to neutralizing antibodies, HeLa cells will be plated in a 96-well
plate for 24h in media. Serum collected from mice vaccinated against adenoviruses will
be added in triplicated to the three different sample populations. Another set of samples
will be cultured without the neutralizing serum. The cells will be cultured for 36 hours
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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
prior fluorescence quantification. TECAN measurements will quantify the transfection
ability of the liposomes by measuring the green fluorescence expression in the wells.
TECAN samples will be prepared by removing the media from the culture plate, rinsing
it two times with PBS and resuspending the cells in 100 L of PBS. The plate will be
placed in the TECAN instrument and fluorescence measurements will be taken. After
the TECAN readings, fluorescence microscopy images will be taken to visually assess
the green fluorescence expression.
Purification of Liposomes
Narrowing the range of size of the liposome particles is important so that one can
deliver a homogenous treatment. Liposome nanoparticles that are too large could
potentially contain multiple adenoviruses instead of the proposed one advenovirus per
liposome nanoparticle. Liposome particles that are too small might not be able to
properly encapsulate an adenovirus. Thus, it is important to narrow down the range of
size of the liposome particles so that the delivered treatment is as desired. Pressure
filtration syringes are inexpensive and size exclusion chromatography is extremely
accurate with proper calibration.
The liposome sample will be added to the filtration syringe and pressure will be applied
slowly to allow proper filtration of the sample. The filtered sample will be collected in a
separate container. By employing a pressure filtration syringe to filter out overly large
liposome nanoparticles (>300nm), we are left with a sample of liposome nanoparticles
under 300nm. This sample will be loaded into a size exclusion chromatography which
will elute the larger particles first. When eluting particles are around 200nm, the
machine will be turned off. The product you receive will contain liposome nanoparticles
200-300nm in size. Data will be presented on a plot showing absorbance versus time.
The plot will present a distribution of liposome nanoparticles eluted and how well the
nanoparticles are separated can be measured using peak separation and peak to valley
ratios. A good separation will indicate that we have indeed narrowed our range of size
of liposomal nanoparticles.
Purifying the sample of any free-floating adenoviruses in solution is crucial because we
dont want free-floating adenoviruses to enter the patients body. The anionic liposomes
with specific receptors will enable only targeted tissue to be exposed to the
adenoviruses. Removal of free-floating adenoviruses will reduce the probability of an
immune response when the liposomes are administered in vivo. Immunopreceipitation
is a method that will allow us to separate the free-floating viruses without harming the
encapsulated complexes. Immunopreceipitation is a high yield method that also
provides a high purity product. The immunoprecipitation will consist of targeting
adenoviral surface proteins with an antibody and subsequent binding of the antibodies
with magnetic beads. The bound magnetic beads to the free-floating adenoviruses will
be removed by applying magnetic field to the sample and removing the supernatant. As
an alternative method to remove the non-encapsulated adenoviruses, magneticactivated cell sorting (MACS) can be utilized. A limitation of this approach is that the

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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
addition of antibodies and magnetic beads could alter the surface properties of the
liposomes.
The immunoprecipitation step will be done by adding Anti-Hexon IgG to the samples
and incubate them for an hour at 4C. Next, magnetic protein G beads will be added
and incubated for an hour at 4C. Magnetic field will be applied to collect all the
magnetic beads bound to the antibody-virus complexes. As an alternative, the sample
can be loaded to the MACS instrument and allowed to be sorted into two populations:
samples containing the magnetic beads (free-floating viruses) and samples that dont
contain the magnetic beads (encapsulated viruses).
In vivo mouse model testing
After confirming the efficacy of the adenoviral liposomes through in vitro testing with
CAR-negative and CAR-positive cells as well as purifying the liposomes to the correct
size (200 to 300 nm), steps must be taking to examine the effects of the treatment in
mice. While the in vitro results will confirm the ability of the anionic liposome bearing the
adenovirus to penetrate and transfect the cells of interest, further in vivo testing will be
required in order to support the aforementioned data. The natural biological
environment provided by the multiple organ systems within the mice models will present
many complexities not seen in the in vitro tests. The liposomes will be subject to many
different cell types (neutrophils, lymphocites, macrophages, etc.) and antibodies as the
circulatory system transports the molecules throughout the entire organism. The
complex interactions proposed by the environment will need to be examined in detail,
and this is achieved injecting the liposomes into the tumor bearing mice models,
histologically analyzing the different organs and tissues of interest, and examining the
tumor size over time.
Transgenic lung tumor bearing mice will be purchased from the Jackson Laboratory, a
company which specializes in the development and selling of genetically manipulated
mice. In order to initiate the activation of tumor development within the mice, intranasal
infection with adenoviral Cre will result in the development of high frequency lung
tumors after six months. The Cre recombinant adenovirus expresses Cre recombinase,
an enzyme that regulates the unwinding of DNA between loxP sites on the DNA. LoxP
is a 34 base pair sequence of DNA which flanks the DNA sequence of interest. The
combination of Cre recombinase and the loxP sites will allow for site-specific
recombination of DNA or the deletion of entire segments of DNA to create the mutation
necessary for lung tumor genesis.
After allowing the transgenic mice to develop the lung tumors for six months,
intravenous tail vein injections of the adenovirus containing liposomes will begin. The
control groups for this study will include a saline solution, anionic liposomes containing
the adenovirus as well as folate as targeting ligands, anionic liposomes with the folate
ligands but no adenovirus, and anionic liposomes with adenovirus but no ligands.
Cancer cells overexpress folate receptors so using folate as a targeting agent for the
anionic liposome will allow the particles to localize around and within the tumor. Prior to
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Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
injection, each mouse is weighed in order to ensure that only up to one percent of the
animals body weight in volume is administered per injection, and the solutions to be
injected are measured using a pH probe to ensure that they fall within the pH range of
4.5 and 8. To begin the injection procedure, the mice will be grabbed, anesthetized
lightly, and placed in a restraining device which allows for the tail to be exposed. In
order to dilate the tail vein for easier injection, the tail is immersed in warm water for five
to ten minutes. A 26 to 27 gauge needle is selected along with a one or three milliliter
syringe, and a solution containing one of the previously mentioned controls is injected
into the proximal third of the tail.
Because 40 million cells per milliliter is a reasonable concentration for cells injected
intravenously, and the nanoparticles are on a much smaller size scale, this
concentration will be used as a standard when administering the treatment. The
liposomes are on the nanometer scale while the cells are in the micrometer scale, and
this concentration ensures that there will be no clumping of particles upon
administration. The varying nanoparticle treatments will be injected intravenously every
three days for the next eight weeks, and histological as well as macroscopic analysis
will help confirm the hypothesis that the encapsulated adenovirus within the ligand
targeting anionic liposome will reduce tumor size. Treating the mice every three days
should ensure that enough nanoparticles are reaching the tumor.
At weeks two, four, six, and eight the mice will be euthanized in order to
macroscopically analyze the tumors and histologically analyze other surrounding organs
and tissues. Because there are four control groups and four euthanizing periods,
sixteen mice will be required to complete the experiment, but thirty-two mice will be
used in order to add more validity and statistical strength to the results. Euthanizing the
mice will occur by performing an intraperitoneal injection of sodium pentobarbital into
the body cavity. 200 mg of sodium pentobarbital per kg of mice will be administered at a
concentration of 60 mg/mL, and the lungs as well as the heart, liver, kidneys, intestines,
brain, spleen, and pancreas will be excised shortly after. This drug is highly toxic, and
care must be taken to make sure that it is not accidentally ingested by researchers. The
use of gloves should prevent this. The other organs besides the lungs will be allowed to
soak on a paper towel and quickly placed in a container containing dry ice to keep the
organs cool.
Using a scalpel and other surgical tools, the mouse equivalent of a pneumonectomy will
be performed in order to completely remove the lungs, and very precise surgical
techniques will be applied to separate the tumor from the lungs. The tumors are then
measured using calipers to estimate the volume, and this is compared to another
measurement: the displacement of water by the tumor. The tumors are also weighed on
a standard lab scale to determine the mass and weight. If the tumors in the mice with
liposomes containing folate and oncolytic adenovirus significantly decrease in size over
the course of eight weeks, this will show that this treatment is more effective compared
to the other controls in vivo.

12

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
To begin the histological evaluation, 4% formaldehyde in phosphate buffered saline is
applied to all of the excised tissues in order to prevent the tissue degradation and
maintain the structure of sub-cellular components. After fixing, the tissue is dehydrated
by transferring the samples to more and more concentrated solutions of ethanol, and
molten paraffin wax is applied to infiltrate the tissue, harden, and allow for the cutting of
very thin sections. Once the wax hardens, the tissues are sliced into 4 micrometer thick
sections using a steel knife mounted on a microtome. The sections are stained with
Haematoxylin and Eosin to visualize the integrity of the nucleus and cytoplasm of the
cells, respectively. Other staining mechanisms will involve the use of primary antibodies
that locate and bind to the anionic liposomes in combination with fluorescent secondary
antibodies. The use of a fluorescence microscope will allow for this visualization, and
this in turn will allow researchers to visualize where the nanoparticles localized in the
mice.
E) Animal Subjects
Human subjects will not be used in this series of experiments because clinical trials will
not reasonably fit within the nine month time frame. As a result, the in vivo experiments
will be centered on the use of transgenic mice ordered from the Jackson Laboratory.
The mice ordered will be of stock number 008179 and carry a point mutation (G12D) in
Kras gene whose expression is blocked by a loxP flanked stop codon. The interaction
with the Cre-recombinase will excise this stop codon and allow said DNA segment to
express the gene for lung tumor genesis. The mouse will be over eight months old
because 6 months are required for the tumor to develop before the treatment is applied,
and 16 males and 16 females will be chosen to allow for diversity as well as statistical
significance with respect to the in vivo studies mentioned earlier. The strain of mice is
the following: B6.129S4-Krastm4Tyj/J. Mice will be used for these studies opposed to
other animals because mice are small, easy to handle, and provide a sufficient
biological model for the treatment plan. Larger animal models would require greater
funds and resources to maintain and would make it difficult to test for many controls.
With regards to veterinary care of the mice, measures will be taken to ensure that
discomfort, distress, and pain are limited to the greatest extent possible for mice
undergoing tumor development. While the complete elimination of the aforementioned
factors is not possible simply because tumor development is not a pleasant process, the
mice will be subject to high quality veterinary care as well as low stress conditions. The
mice will be exposed to a 14 hour light followed by 10 hour dark cycle, and researchers
will not disturb the mice during the 10 hour dark cycle to minimize stress on the animals.
The mice will be placed in separate cages at temperatures of 18 to 23 degrees Celsius
at 40% humidity. Clean water will be available at all times, and the mice will be fed diets
consisting of less than ten percent fat. The mice will be handled as gently as possible,
and noise and vibrations will be limited to decrease stress as well. New gloves will be
used when dealing with different mice to minimize the exposure to different scents,
which can also help in alleviating stress.

13

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
Before intravenous tail vein injections of the several controls, the mice will be restrained
in a device that limits their movement and exposes the tail. This device is part of
standard practice laboratory procedures, and anesthesia will limit the pain felt by the
animal as well. The anesthesia will be injected into the body cavities (intraperitoneal
injection) of the mice, and the combination of ketamine and xylazine will serve to sedate
the animal, reduce stress, and make the IV injection process easier. The anesthetic
doses will be between 50 to 100 mg/kg and 7 to 20 mg/kg of ketamine and xylazine,
respectively. The euthanasia process will be applied before harvesting the mice organs,
and more details regarding the use of sodium pentobarbital are elucidated in the
previous section. The methods described above are consistent with the
recommendations of the Panel of Euthanasia of the American Veterinary Medical
Association, and following these standards will secure the comfort of the mice used in
this experimental model.
F) References
[1] Ferguson, Mark S., Lemoine, Nicholas R., and Yaohe,Wang. "Systemic Delivery of Oncolytic
Viruses: Hopes and Hurdles." Advances in Virology 2012.10 (2012): 1-14.
[2] Chiocca, E. Antonio. "Oncolytic Viruses." Nature Publishing Group 2.1 (2002): 938-961.
[3] Kelly, Elizabeth and Russell, Stephen J. "History of Oncolytic Viruses: Genesis to Genetic
Engineering." Molecular Therapy 15.4 (2007): 651-659.
[4] Bertil Hille Ion channels of excitable membranes, 3rd ed., Sinauer Associates, Sunderland,
MA (2001)
[5] Tseng, Li-Ping, Liang, Hong-Jen, Chung, Tze-Wen, Huang, Yi-You, Liu, and Der-Zen.
"Liposomes Incorporated with Cholesterol for Drug Release Triggered by Magnetic
Field."Journal of Medical and Biological Engineering 27.1 (2007): 29-34.
[6] Wang, Rongrong, Xiao, Renzhong, Zeng, Zhaowu, Xu, Lili and Wang, Junjie. "Application of
poly(ethylene glycol)distearoylphosphatidylethanolamine (PEG-DSPE) block copolymers and
their derivatives as nanomaterials in drug delivery." Journal of Nanomedicine 7.1 (2012): 41854198.
[7] Yotnda, Patricia, Hicks, A. R. Davis, Templeton, N. S. and Benner, M. K. "Liposomal
Enhancement of the Antitumor Activity of Conditionally Replication-Competent Adenoviral
Plasmids." Molecular Therapy 9.4 (2004): 489-495.
[8] Zhong, Zhiron, Yu Wan, Jianfeng Han, Sanjun Shi, Zhiron Zhang, and Xun Sun.
"Improvement of Adenoviral Vector-mediated Gene Transfer to Airway Epithelia by Folatemodified Anionic Liposomes." International Journal of Nanomedicine (2011): 1083-093. Web. 3
May 2013.
[9] Christina R. Miller, Bruce Bondurant, Shannon D. McLean, Kathy A. McGovern, and David F.
OBrien. Liposome-Cell Interactions in Vitro: Effect of Liposome Surface Charge on the Binding
and Endocytosis of Conventional and Sterically Stabilized Liposomes. Biochemistry (1998), 37,
12875-12883.
[10] Sunita. The Cancer Market Outlook To 2014: Competitive Landscape, Market Size,
Pipeline Analysis and Growth Opportunities Now Available on ReportsandReports. <
http://www.prnewswire.com/news-releases/the-cancer-market-outlook-to-2014-competitivelandscape-market-size-pipeline-analysis-and-growth-opportunities-now-available-onreportsandreports-87326377.html>.
[11] Dua, J.S., A.C. Rana, and A.K. Bhandari. "Liposome: Methods of Preparation and
Applications." International Journal of Pharmaceutical Studies and Research 3.2 (2012): 14-20.
Print.

14

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
[12] Wiley. Liposomes in Nanomedicine. N.p. Web.
<http://books.google.com/books?id=stfipzoxvHwC&printsec=frontcover#v=onepage&q&f=false>
.
[13] Juarez, Pepe. "Electronic Supplementary Material (ESI) for Integrative Biology." The Royal
Society of Chemistry 2012 (n.d.): n. pag. Web.
<http://www.rsc.org/suppdata/ib/c2/c2ib20122d/c2ib20122d.pdf>.
[14] Dai, Yeling. "Introduction to Dynamic Light Scattering (DLS)." N.p., 30 Apr. 2008. Web.
<http://x-ray.ucsd.edu/mediawiki/images/0/03/Dynamic_light_scattering_group_apr30.pdf>.
[15] Erbse, Annette. "Basic Protocol for Dynamic Light Scattering" 13 June 2013
<http://chem.colorado.edu/biochemcore/images/Basic_protocol_for_DLS_JMW_corrected.pdf>.
[16] "Basic SEM Protocol" The Ohio State University Campus Microscopy and Imaging Facility.
The Ohio State University. 4 June 2013 <http://cmif.osu.edu/418.cfm>.
[17] "The Institutional Animal Care and Use Committee" UCSF Medical Center. UCSF. 30 May
2013 <http://iacuc.ucsf.edu/Policies/awSPtailveinjection.asp>.
[18] "IV Injection Recommendations for mice" Frederick National Laboratory for Cancer
Research. 5 June 2013 <http://ncifrederick.cancer.gov/rtp/lasp/intra/acuc/beth/IV-InjectionRecommendations-mice.pdf>.
[19] "Intravenous Injection in the Mouse" Procedures with Care. Newcastle University. 12 June
2013 <http://www.procedureswithcare.org.uk/intravenous-injection-in-the-mouse/>.
[20] "Featured JAX mice for cancer research" Jaxmice. The Jackson Laboratory. 12 June 2013
<http://jaxmice.jax.org/cancer/featured.html#lung>.
[21] "Cre Recombinase Adenovirus" Vectorbiolabs. Vector Biolabs the Gene Delivery Company.
10 June 2013 <http://www.vectorbiolabs.com/Recombinant-Adenovirus/1045/Ad-CMV-Cre/CreRecombinase>.
[22] "Standard Euthanasia Guidelines for Rodents" The Institutional Animal Care and Use
Committee. UCSF Medical Center. 8 June 2013
<http://www.iacuc.ucsf.edu/Policies/awGlEuthR.asp>.
[23] "Mouse Organ Harvest Protocol" Cancer UCSD Training. UCSD .
<http://cancer.ucsd.edu/research-training/sharedresources/Documents/mouse_organs_for_IHC_WTko.pdf>.
[24] "Mouse Room Conditions" Jaxmice. The Jackson Laboratory. 7 June 2013
<http://jaxmice.jax.org/support/husbandry/room-conditions.html>.
[25] "Ketamine-Xylazine Combination for Rodent Anesthesia" Laboratory Animal Sciences
Program. NCI at Frederick. 2 June 2013
<http://ncifrederick.cancer.gov/rtp/lasp/intra/acuc/beth/KetamineXylazine.asp>.
[26] Meuwissen, R. "Mouse Models for Human Lung Cancer." Genes & Development 19.6
(2005): 643-64. Print.
[27] Cheon, Dong-Joo, and Sandra Orsulic. "Mouse Models of Cancer." Annual Review of
Pathology: Mechanisms of Disease (2011): 95-119. Web. 7 June 2013.
[28] Ho, V. M., B. E. Schaffer, A. N. Karnezis, K. S. Park, and J. Sage. "The Retinoblastoma
Gene Rb and Its Family Member P130 Suppress Lung Adenocarcinoma Induced by Oncogenic
K-Ras." Oncdogene 28.10 (2009): 1393-399. Print.

Mine
-[1]

http://www.technicaljournalsonline.com/ijpsr/VOL%20III/IJPSR%20VOL%20III%20ISSUE%2
0II%20APRIL%20JUNE%202012/Article%204%20April%20June%202012.pdf
-[2]
http://books.google.com/books?id=stfipzoxvHwC&printsec=frontcover#v=onepage&q&f=false
15

Group #9: Anionic liposomal encapsulation of oncolytic adenovirus


Nicolas Etcheverry (A08662261), Vanessa Herrera (A09202452)
-[3] http://www.rsc.org/suppdata/ib/c2/c2ib20122d/c2ib20122d.pdf
-[4] http://x-ray.ucsd.edu/mediawiki/images/0/03/Dynamic_light_scattering_group_apr30.pdf
-[5]
http://chem.colorado.edu/biochemcore/images/Basic_protocol_for_DLS_JMW_corrected.pdf
-[6] http://cmif.osu.edu/418.cfm
Nicos sources
-[1]
http://iacuc.ucsf.edu/Policies/awSPtailveinjection.asp
-[2]
http://ncifrederick.cancer.gov/rtp/lasp/intra/acuc/beth/IV-Injection-Recommendations-mice.pdf
-[3]
http://www.procedureswithcare.org.uk/intravenous-injection-in-the-mouse/
-[4]
http://jaxmice.jax.org/cancer/featured.html#lung
-[5]
http://www.vectorbiolabs.com/Recombinant-Adenovirus/1045/Ad-CMV-Cre/Cre-Recombinase
-[6]
http://www.iacuc.ucsf.edu/Policies/awGlEuthR.asp
-[7]
http://cancer.ucsd.edu/research-training/sharedresources/Documents/mouse_organs_for_IHC_WTko.pdf
-[8]
http://jaxmice.jax.org/support/husbandry/room-conditions.html
[9]
http://ncifrederick.cancer.gov/rtp/lasp/intra/acuc/beth/KetamineXylazine.asp

16

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