Magnetic Nanoparticles in the Treatment of Obesity-

Driven Cancers and the Associated Toxicities

Saoirse Morrin

Supervisors: Graham Pidgeon, Maria Santos Martinez,

Oliviero Gobbo 21/06/23
 Oesophageal Adenocarcinoma
A Clinical Challenge
Oesophageal adenocarcinoma (OAC) is a growing problem in developed countries,
corresponding with an increase in BMI
.

5-year survival is poor at just 22%

The standard of care comprises of neoadjuvant chemo-radiotherapy, which may be followed by

resection

Fewer than one third of patients respond to treatment

New strategies are needed

Trinity College Dublin, The University of Dublin 2

Oesophageal Adenocarcinoma and Obesity
Two Clinical Challenges Interlinked

Trinity College Dublin, The University of Dublin 3

Nanomedicine
An Emerging Strategy in Cancer Treatment

Trinity College Dublin, The University of Dublin 4

Magnetic Nanoparticles
The Answer to both Cancer and Obesity?

Superparamagnetic iron oxide nanoparticles have a maghemite (Fe3O4) core, but each
have a different coating in order to be biologically compatible

Coatings play a critical role in cellular uptake, function and subsequent toxicities and
different coatings can confer different functions

Coatings

PAA – Polyacrylic Acid - negatively charged

DMSA – Meso-2,3-DiMercaptoSuccinic Acid - negatively charged

AEPA – AminoEthylPhosphonic Acid - positively charged

5
Alternating Magnetic Field Apparatus

Petri Dish

Rotating
Platform
Motor

Magnet

6
 Hypothesis and Aims
Overarching Hypothesis
Non-targeted iron MNPs could be a therapeutic
option to target oesophageal cancer and / or
adipose tissue by inducing magneto-
mechanical stress while inducing minimal
toxicities in healthy tissue.
Aims
1. To characterise the various coatings of MNP
based on size and charge
2. To determine the cytotoxic effects of MNPs of
various coatings on components of blood
3. To determine the dose required to induce cell
death in oesophageal cancer cells
4. To determine the potential of targeting visceral
adipose tissue fragments using MNPs
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Aim 1

1. To characterise the various coatings of MNP based on size and charge

2. To determine the cytotoxic effects of MNPs of various coatings on components of

blood

3. To determine the dose required to induce cell death in oesophageal cancer cells

4. To determine the potential of targeting visceral adipose tissue fragments using MNPs

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Characterisation of MNPs
Malvern Zetasizer

Basis of Zetasizer
Zetasizer illuminates the particle using laser
light. Dynamic light scattering (DLS) can be used
to quantify particle size and charge based on
the intensity of fluctuations in scattered light.
Larger particles move more slowly, creating
slower variations in intensity.

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10
Aim 2

1. To characterise the various coatings of MNP based on size and charge

2. To determine the cytotoxic effects of MNPs of various coatings on components of

blood

3. To determine the dose required to induce cell death in oesophageal cancer cells

4. To determine the potential of targeting visceral adipose tissue fragments using MNPs

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Protein Corona
A barrier to MNP efficacy?

In each blood experiment, the protein corona will be emulated either by

foetal bovine serum or platelet poor plasma.

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 Red Blood Cell Viability
Are MNPs toxic to RBCs?

Aims of Experiment Method

1. RAW 264.7 cells are seeded with DMEM

1. To determine the level of cytotoxic effects of
PAA MNPs on RBCs at various concentrations
2.To determine the concentration of PAA to use
with MF apparatus
with 10% FBS, media is changed after
24hrs
2. 24hrs later the cells are treated with
various concentrations of PAA, control is
untreated

3. 24hrs later both live and dead cells are

counted

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Results

No statistical significance was found between the Statistical significance was found between
groups with 10% FBS, 0.5% FBS and no FBS, the control and concentrations of 75 and
indicating little effect of the protein corona on MNP 100 mg/mL, indicating cytotoxicity. Hence,
cytotoxicity we elected to use a conc of 75 for MF.

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 Red Blood Cell Viability with MF
Will an AMF increase cytotoxic effects of MNPs on RBCs?

Aim of Experiment Method

To determine the level of cytotoxic effects of PAA 1. RAW 264.7 cells are seeded with DMEM with
MNPs on RBCs when activated by an alternating 10% FBS into 4 petri dishes, media is changed
magnetic field after 24hrs

2. 24hrs later the cells are treated with various

concentrations of PAA, controls are untreated

3. 24hrs later each petri dish is placed on the MF

apparatus for 30 minutes followed by a 2 hour
incubation followed by another 30 minutes on
the apparatus

4. 24hrs later the cells are counted

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Results

‒ Statistical significance was found between

both controls and both groups with MNPs,
indicating cytotoxicity
‒ Statistical significance (p=0.004) was also
found between the group with MNPs
exposed to MF and the group with MNPs
not exposed to MF, indicating increased
cytotoxicity when exposed to MF

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 Haemolysis of Red Blood Cells
Will various MNPs induce breakdown of RBCs?
Aim of Experiment
To determine the rate of haemolysis induced by
various concentrations of various coatings of
MNPs.
Basis of Experiment
If haemolysis occurs, haemoglobin will be
released, and levels can be read from the
supernatant of the sample.
Method
1. 10mL of whole blood is obtained from a healthy
volunteer, centrifuged and is washed with both saline
and PBS before 1mL of washed RBCs is diluted into
49mL of PBS or 48mL PBS and 1mL PPP
2. The RBCs are seeded into a 96 well plate and treated
with various concentrations of each MNP. The negative
control is untreated while the positive control is
treated with 20% Triton
3. The plate is incubated for 1 hour and is then
centrifuged
4. The supernatant is removed and placed into a new
plate to be read at 450nm
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Results

Statistical significance was found No such statistical significance was

between the negative control and PAA of found when PPP was present,
concentrations 50 mg/mL and higher, suggesting a protective role of the
when no PPP is present, indicating protein corona against haemolysis.
haemolysis.

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 Platelet Aggregation
Will MNPs induce platelet aggregation?

Basis of Light Transmission Aggregometry

The cuvette containing the MNPs, and platelets
is placed between a light source and a
photocell. If aggregation occurs the platelets
will absorb less light, transmission increases
and will be detected by the photocell.

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Raw Data from Platelet Aggregation Profiler PAP-8E

Positive Control - Collagen PAA, DMSA, AEPA 25,50,75,100 +

Negative Control (PRP only)
%

A
g
g
r
e
g
a
ti
o
n
Time (mins) Time (mins)

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Results

No statistical significance was found between

any concentration of any MNP and the negative
control.
This indicates little platelet aggregation was
induced by any MNP.
To confirm this a VEGF ELISA was performed on
each supernatant. VEGF is released when
platelets aggregate. No increase in VEGF was
found, therefore confirming a lack of platelet
aggregation.

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Summary
Aim 2. To determine the cytotoxic effects of MNPs of various coatings on components of
blood

1. Exposure to PAA MNPs for 24hrs demonstrates a decrease in cell viability, with
statistically significant results at concentrations 75 mg/mL and above.
2. This cytotoxicity is increased when exposed to AMF, with statistically significant results
3. PAA MNPs demonstrate statistically significant haemolysis at concentrations 50 mg/mL
and above without PPP, but no statistically significant haemolysis with PPP.
4. None of the MNPs demonstrated statistically significant platelet aggregation at any
concentration analysed.

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Aim 3

1. To characterise the various coatings of MNP based on size and charge

2. To determine the cytotoxic effects of MNPs of various coatings on components of blood

3. To determine the dose required to induce cell death in oesophageal cancer cells

4. To determine the potential of targeting visceral adipose tissue fragments using MNPs

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 MTT Assay
Will various coatings of MNPs induce OE33 cell death?

Method

Basis of Assay 1. 100microL of MTT reagent is added to

This assay is based on the cleavage of the
tetrazolium ring of MTT by dehydrogenases in 2.
active mitochondria of living cells as an
estimate of viable cell number.
3.
cultured cells and incubated for 4 hours
The contents of each well is removed and
replaced with DMSO and is incubated for
10 minutes
Colorimetric results are obtained at
560nm

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Results

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 BrDU Assay
Will various coatings of MNPs reduce cell viability?

Basis of Assay
BrDU is incorporated into de-novo synthesized DNA as
a substitute for thymidine to label proliferating and
daughter cells.
Method
1. 10microM of BrDU labelling solution is added
to cultured cells and incubated for 4 hours
before the contents of the wells are removed
2. Cells are fixed with FixDenat for 30 minutes
3. Anti-BrDU-POD working solution is added to
each well for 2 hours before 3 washes
4. Substrate solution is added to each well
5. Colorimetric changes are read at 450nm

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Results

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Aim 4

1. To characterise the various coatings of MNP based on size and charge

2. To determine the cytotoxic effects of MNPs of various coatings on components of blood

3. To determine the dose required to induce cell death in oesophageal cancer cells

4. To determine the potential of targeting visceral adipose tissue fragments using MNPs

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 AEPA Co-Culture with Fat Explants for VEGF ELISA
Will co-culture of AEPA with fat explants result in a cytokine burst?

Processing of Omentum Method

1. 50 mg/mL conc of AEPA is added to the 2
leftmost wells and is incubated for a further
1. 500g of visceral fat is placed into 4 wells of a 12 24Hrs
well plate
2. The plate is placed on the AMF device for 30
2. 1mL of media (M199 + gentamicin) is added to minutes before a 2-hour incubation and a further
the well 30 minutes on the device
3. The fat is diced as small as possible using a 3. 24Hrs later the supernatant is collected
scissors
4. ELISA is performed with suitable reagents and
4. The tissue is incubated for 24Hrs incubation periods and a standard curve is
generated

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VEGF ELISA Results

Statistically significant results

were. obtained when VEGF
expression was compared
between the control and both
groups (p=0.013). This
R2= 0.9868 indicates increased VEGF
production, suggesting
induction of a cytokine burst
by MNPs

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Imaging of Internalisation of AEPA into Adipocytes

Adipocytes without AEPA Adipocytes with AEPA exposed

to AMF

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 Summary
Aim 4.

‒ Both MNPs with and without exposure to AMF, when co-cultured with fat tissue
explants demonstrated a statistically significant increase in VEGF, suggesting
induction of a cytokine burst

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 Future Perspectives

Short Term Long Term

‒ Optimisation of the alternating magnetic ‒ in vivo studies
field device
‒ Expand to further cell lines, including
splenic and hepatic tissue
‒ Clonogenic Assays
‒ Further investigation into immune cells
and/or cytokines involved in cytokine burst
‒ Imaging of internalization into cells

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 Acknowledgements

Graham Pidgeon
Maria Santos Martinez
Oliviero Gobbo
Thank You Tianyi Chen
The Biobanking Team
Fiona O’Connell
The Patients of SJH