Learning Objectives: Understand convection-enhanced delivery and its implication for brain tumour treatment Understand how gold nanoparticles can be used to construct radiation nanomedicine Learn how to evaluate the safety, toxicity, and effectiveness of radiation nanomedicines Overview: Glioblastoma is a devastatingly aggressive type of brain tumour with a low median, and 5-year survival that has lacked new treatment options, in part due to the inability of therapeutic agents to cross the blood-brain barrier. Convection Enhanced Delivery (CED), a clinical neurosurgical strategy has been used to locoregionally deliver various therapeutic agents within the brain. Radiotherapeutic agents, such as 177Lu-labeled gold nanoparticles (177Lu-AuNP), hold promise for treatment of glioblastoma when administered by CED. Intratumoural injections of 177Lu-AuNP administered by CED was evaluated in an orthotopic xenograft mouse model of glioblastoma. SPECT/CT and biodistribution studies were used to evaluate the fate of the 177Lu-AuNP after injection. These results were used to estimate organ radiation absorbed doses. Normal tissue toxicity was evaluated to confirm the safety of the injections. Magnetic resonance imaging and bioluminescence imaging were used to monitor tumour growth after administration of 177Lu-AuNP, and median survival was estimated.

1. Evaluating Intracerebral Injections of Radiation
Nanomedicine in a Preclinical Mouse Model of
Glioblastoma
Constantine Georgiou
PhD Candidate, Department of Pharmaceutical Sciences
University of Toronto

2. Outline
1. Introduction to Glioblastoma (GBM) and treatment
2. What is radiation nanomedicine?
3. Animal models, intracerebral injections, methods
4. Biodistribution, SPECT/CT imaging, dosimetry, toxicity
5. Therapeutic evaluation
6. Future directions
2

3. Glioblastoma (GBM)
2/10 Canadians diagnosed
with any brain tumour will
survive 5 years
<1/10 Canadians
diagnosed with GBM
will survive 5 years
• GBM is the most aggressive and most common malignant brain tumour
• Classified as a high grade (IV) astrocytoma
• While rare compared to other cancers, GBM is always fatal
• GBM incidence: 3-4 per 100,000
• GBM median OS: 12-15 months
Glioblastomas
56.60%
Diffuse
astrocytomas
Glioma
malignant
Ependymal
tumors
Anaplastic
astrocytomas
Oligodendrogliomas
Pilocytic
astrocytomas
Oligoastrocytic
tumors
All others
gliomas
3
Background Rationale Results Future Directions Conclusion
Louis, D., et al. Acta Neuropathologica (2016)

4. GBM Standard-of-Care
Background Rationale Results Future Directions Conclusion
Surgical
Resection
60 Gy External
Beam
Radiotherapy
Temozolomide
Chemotherapy
4

5. Treatment Challenges – Residual Disease
The BBB prevents the
majority of chemotherapies
from being effective
Molecular resistance
to Temozolomide
chemotherapy
Complete resection
is impossible
Limited to 60 Gy by external
beam radiotherapy
5
Background Rationale Results Future Directions Conclusion
1. Treatment fails to eliminate residual disease
2. Recurrence occurs within 2-4 cm of the
original tumour
3. No standard treatment for recurrent
disease
What strategies are
available to solve this
problem?

6. Convection Enhanced Delivery (CED)
• Most therapeutic agents do not
reach effective concentrations
after oral or I.V. administration
• CED catheters are inserted into
the tumour region
• External infusion pump creates a
pressure gradient that infuses
the therapeutic agent
• Compatible with wide range of
therapeutic agents
6
Mehta, A.M., et al. Neurotherapeutics (2017)
Background Rationale Results Future Directions Conclusion

7. Background Rationale Results Future Directions Conclusion
Selecting a Therapeutic Agent for CED
7
AuNP Chelator Radionuclide
Radiolabeled AuNP

8. Functionalizing Gold Nanoparticles (AuNP)
• Functionalization is a key ability of AuNPs
• AuNP drug delivery alters the PK of the
therapeutic agent
• Compatible with a wide variety of
therapeutic molecules
• Radionuclides are uniquely positioned
for treating GBM residual disease
• Cancer has reduced capability to repair DNA
damage caused by ionizing radiation
• Generates a predictable therapeutic field
8
Her, S., Jaffray, D.A., Allen, C. Adv. Drug Deliv. Rev. 2017
Inside Particle
Range
Outside
Particle Range
Background Rationale Results Future Directions Conclusion

9. AuNP Functionalization – Metal Chelating Polymer
• Coat AuNP surface with di-block
metal chelating polymer (MCP)
• Section 1: PEG 2kDa
• Increases stability, reduces
aggregation and MPS uptake
• Section 2: poly-glutamine peptide
with 8 pendant DOTA
• Chelates large amounts of activity
• Section 3: poly-glutamine peptide
with 4 pendant Lipoic Acid groups
• High number of Au-S bonds
increases stability
1 2 3 Dr. Mitch Winnik
Department of
Chemistry
U of T
9
Background Rationale Results Future Directions Conclusion

10. Radiation Nanomedicine – Radionuclide
10
Pouget, J.P., et al. Nat. Rev. Clin. Oncol. (2011)
β- α
Auger Electron
Physical
Parameter
β- Particle α Particle
Auger
Electron (AE)
Energy 0.05 – 2 MeV 5 – 9 MeV <25 keV
Range in
Tissue
mm – cm
pathlength
Many cell
diameters
μm – mm
pathlength
Several cell
diameters
nm – µm
pathlength
≤ 1 cell
diameter
Linear Energy
Transfer
0.1 – 1.0
keV/µm
50 -230
keV/μm
4 – 26
keV/µm
Best Suited
For:
Small to
medium
tumours
Small volume
metastases
Single cells,
micro-
metastases
Radionuclide 177Lu 225Ac 111In
Background Rationale Results Future Directions Conclusion

11. Radiation Nanomedicine – Hypothesis
CED of AuNPs radiolabeled with 177Lu will be effective in controlling GBM recurrence
AuNP
Metal Chelating
Polymer (MCP) 177Lu 177Lu-MCP-AuNP
Intraoperative
Administration for
Residual Tumour
11
Background Rationale Results Future Directions Conclusion

12. Animal Model and Experiments
Inoculate NRG mouse
with U251-Luc Human
GBM cells (2x105
cells/mouse)
Inject with 5 µL of
177Lu-AuNP or
control
SPECT/CT +
MRI + BLI
12
Biodistribution
Toxicity
Therapy
Background Rationale Results Future Directions Conclusion

13. Single Photon Emission Computed
Tomography (SPECT)
13
Gamma Photon
Emitting Radionuclide
(e.g. 177Lu)
γ
SPECT/CT
Background Rationale Results Future Directions Conclusion

14. Biodistribution – microSPECT/CT Imaging
14
177
Lu-MCP-AuNP
177
Lu-MCP
Day 0 Day 7 Day 14 Day 21
Day 0 Day 1 Day 2 Day 3
Representative
1 MBq 177Lu
CED injection,
not decay
corrected
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

15. Biodistribution – Whole Body Retention
15
0 5 10 15 20
0
25
50
75
100
125
Whole Body Retention After Intracranial Injection
Days Post Injection
%ID
(Decay
Corrected)
177
Lu-MCP
177
Lu-AuNP
*
Background Rationale Results Future Directions Conclusion
Dose Calibrator

16. Biodistribution & Radiation Dosimetry
• Measure cumulative
radioactivity (Ã) in
critical organs from
1 h – 14 d
• Obtain published S
values
• Estimate absorbed
dose per organ using
MIRD equation
16
𝐷 = 𝐴 × 𝑆
1
2
3
4
6
7
A B
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

17. Biodistribution of 177Lu-AuNP
17
Right
Left
Cerebellum
B
l
o
o
d
H
e
a
r
t
L
u
n
g
L
i
v
e
r
S
p
l
e
e
n
P
a
n
c
r
e
a
s
S
t
o
m
a
c
h
S
m
a
l
l
i
n
t
e
s
t
i
n
e
K
i
d
n
e
y
s
M
u
s
c
l
e
B
o
n
e
S
k
i
n
B
r
a
i
n
(
R
i
g
h
t
)
B
r
a
i
n
(
L
e
f
t
)
B
r
a
i
n
(
C
e
r
e
b
e
l
l
u
m
)
0
2
4
6
8
200
300
400
500
177
Lu-AuNP Biodistribution
%ID/g
1 HPI
24 HPI
72 HPI
168 HPI
336 HPI
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

18. Radiation Dose Estimates of 177Lu-AuNP
• Intratumoural injections in the
brain stay localized at injection
site
• Highest dose delivery to tumour
• Little irradiation of other brain
regions
• Negligible dose to peripheral
organs
18
Organ/Region Absorbed Dose (Gy)
Heart 0.08 ± 0.01
Lungs 0.06 ± 0.01
Liver 0.15 ± 0.03
Spleen 0.22 ± 0.05
Pancreas 0.07 ± 0.02
Stomach 0.09 ± 0.02
Intestine 0.05 ± 0.01
Kidneys 0.08 ± 0.01
Carcass 0.03 ± 0.01
Whole Brain 16.2 ± 5.8
Cerebellum 0.2 ± 0.1
Left Hemisphere (non-tumour bearing) 0.3 ± 0.1
Right Hemisphere (excluding tumour) 6.4 ± 3.3
Tumour 599 ± 311
Background Rationale Results Future Directions Conclusion

19. Toxicity Evaluation
A
L
T
(
U
/
L
)
C
R
E
(
u
m
o
l
/
L
)
G
L
U
(
m
m
o
l
/
L
)
T
P
(
g
/
L
)
W
B
C
(
1
0
9
/
L
)
R
B
C
(
1
0
1
2
/
L
)
H
G
B
(
g
/
d
L
)
P
L
T
(
1
0
9
/
L
)
A
L
P
(
U
/
L
)
B
U
N
(
m
m
o
l
/
L
)
H
C
T
(
%
)
0
25
50
75
100
300
400
500
600
Toxicity - Blood Measurements
Control
1.5 MBq 177
Lu-AuNP
1 3 6 8 10 13
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Weight
Time (days)
Body
Weight
Index
(BWI)
Control
1.5 MBq 177
Lu-AuNP
19
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

20. Audience Poll

21. Tumour Growth – Bioluminescence Imaging
21
Mezzanotte, L. et al. Trends Biotechnol. (2017)
Background Rationale Results Future Directions Conclusion

22. Tumour Growth – T2 Weighted MRI
22
M3 Aspect 1T System
RF
In-Phase
Precession
37%
T2 Time (msecs)
100%
Background Rationale Results Future Directions Conclusion

23. Tumour Growth – BLI
0 7 14 21
0
20
40
60
80
100
Bioluminescent Signal
Days Post Injection
Tumour
Growth
Index
Saline
Non-Radioactive AuNP
177
Lu-AuNP (1.0 MBq)
23
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

24. Tumour Growth – MRI & Histology
Saline Control
Non-Radioactive
AuNP
177Lu-AuNP
MRI 4 Weeks Post Treatment
S
a
l
i
n
e
N
o
n
-
R
a
d
i
o
a
c
t
i
v
e
A
u
N
P
1
7
7
L
u
-
A
u
N
P
(
1
.
0
M
B
q
)
0
10
20
30
40
50
60
Tumor
Volume
(mm
3
)
A B C
24
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion

25. Tumour Growth – MRI & Histology
Saline Control
Non-Radioactive
AuNP
177Lu-AuNP
A B C
25
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion
T: Residual Tumour
M: Tumour Margin
Contralateral (Left)
Hemisphere

26. Tumour Growth – Long Term Survival
26
Georgiou, C. et al. Mol. Pharm. (2022)
Background Rationale Results Future Directions Conclusion
0
7
1
4
2
1
2
8
3
5
4
2
4
9
5
6
6
3
7
0
7
7
8
4
9
1
9
8
0.7
0.8
0.9
1.0
1.1
1.2
1.3
Body Weight
Days Post Injection
Body
Weight
Index
(BWI)
Saline
Non-Radioactive AuNP
177
Lu-AuNP (1.0 MBq)
0 50 100 150
0
50
100
Kaplan-Meier Curve
Days Post Injection
Percent
Survival
Saline
Non-Radioactive AuNP
177
Lu-AuNP

27. Summary
• Glioblastoma remains difficult to effectively treat
• Convection enhanced delivery can be used to bypass the BBB
• Radiolabeled gold nanoparticles are uniquely suited for CED
• SPECT/CT and biodistribution can be used to track 177Lu-AuNP in vivo
• Local delivery confined to tumour with large radiation absorbed dose
• 177Lu-AuNP did not cause acute toxicity
• Molecular imaging (BLI + MRI) can be used to evaluate tumour
growth
• 177Lu-AuNP was extremely effective in controlling GBM growth
27
Background Rationale Results Future Directions Conclusion

28. Future Directions
Checkpoint Immunotherapy Combination
28
Background Rationale Results Future Directions Conclusion

29. Immune System Activation in Cancer
29
Background Rationale Results Future Directions Conclusion

30. PD-1 Immune Checkpoint
30
Background Rationale Results Future Directions Conclusion

31. Anti-PD-1 Immune Checkpoint Inhibitor
31
Background Rationale Results Future Directions Conclusion

32. 32
Bernstein, M.B., et. al. Nat. Rev. Clin. Oncol. (2016)
Background Rationale Results Future Directions Conclusion
Isolate Tumour
Infiltrating
Immune Cells
Collect Whole
Brain from
Treated Mice
Stain and Sort
Immune Cells
(FACS)

33. Acknowledgements
Reilly Lab
Dr. Zhongli Cai
Dr. Conrad Chan
Valerie Facca
Rella Liu
Misaki Kondo
Felix Ho
Madeline Brown
Stephanie Borlase
Rutka Lab
Carlyn Figueiredo
Supervisor
Dr. Raymond Reilly
Committee Members
Dr. Christine Allen
Dr. James Rutka
Dr. Mitchell Winnik
STTARR
Teesha Komal
Deborah Scollard
CPO
Dr. Azza Al-Mahrouki
Scintica
Tonya Coulthard
33

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