Cancer-Translational Nanotechnology Training (Cancer-TNT) Presentation - December 5, 2017 - Stanford Medicine
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Cancer-Translational Nanotechnology
Training (Cancer-TNT) Presentation
T R AV I S S H A F F E R , S A N J I V S A M G A M B H I R
D E PA R T M E N T O F R A D I O L O G Y, S TA N F O R D U N I V E R S I T Y
December 5, 2017
Upscaling and improving Raman
nanoparticles for large animal
studies
Imaging Natural Killer Cells via the
NKp-46 receptor
O
O
Synthesizing new Raman flavors and N
N
OH
coatings for improved in vivo use
HO
N
N
OH
O
O
N
Normalized Raman Spectra
N
S0772
SiCl2
IR-780
500 1000 1500
Raman Shift (cm-1)
3
Biomedical applications of silicated gold Raman
nanoparticles (AuNP)
Andreou C, Kishore
Kircher MF, GambhirSA,
lab.Kircher
NatureMF. JNM 2015.
medicine 2012.
Optimizing the large-scale synthesis of silicated AuNP
Upscaling a citrate-free gold nanoparticle
synthesis for large animal studies
Silica
shell
Raman layer
Exploring silica shell synthetic routes and
their effect on stability
Imaging Gold
metal
Loading imaging agents into the silica
Silica shell for multimodal imaging
shell
IR-780
Wall*, Shaffer*, et al. Theranostics 2017.
Synthesizing monodisperse, citrate-free gold
nanoparticles for large animals
OH
O
HAuCl4 HO O
OH
O-
O Na +
Harmsen, Wall, Kircher, Nature Protocols 2017.
OH
HAuCl4 HO
Wall, Harmsen, et al, Advanced Materials 2017.
Using Tangential flow filtration for higher yields
Average hydrodynamic diameter
AuNP product
• Citrate-free gold surface 60
• Polydispersity index < 0.09 40
nm
• Completed in < 1 hour followed
by overnight ripening 20
• 4L, 0.2 nM AuNP solution 0
1
2
3
4
5
6
7
8
ch
ch
ch
ch
ch
ch
ch
ch
at
at
at
at
at
at
at
at
B
B
B
B
B
B
B
B
Hypothesis: more Si-O-Si bonds in silica shell leads to
increased stability
O O
HO
O
O H 2O Si
Si OH
O
O
O
R1 R2 R1
R4
R2 R3 Si R6
Si
OH R5 H 2O
Si
OH O Si R5
R3
R6
R4
Increasing polarity
Isopropanol Ethanol Methanol
Slower hydrolysis Greater hydrolysis
Faster condensation Slower condensationSynthetic routes for silicating gold nanoparticles
Attach Raman dye, rapid silication of
AuNP with excess TEOS in IPA, no
primer
Harmsen, Wall, Kircher, Nature Protocols 2017.
Attach Raman Slow silication
dye and primer with TEOS in
(MPTMS) EtOHStability of silicated AuNP to etching in 10% saline
Saline Incubation (70 C 30 min)
250 Rapid silication
Diameter (nm)
200
Slow silication
150
Rapid silication
10% saline
100
14 H
50
e
e
lin
iv
at
sa
N
in
C
95
Saline Incubation (RT 14 H) Slow silication
800
Diameter (nm)
600
Slow silication
400
Rapid silication 10% saline
200 14 H
0
e
e
lin
iv
at
sa
N
ur
ho
14A new route for stable, large-scale SERS AuNP
• 1000-fold scale-up a citrate-free gold
nanoparticle synthesis.
• Increased yield and decreased purification
time using tangential flow filtration.
• Investigated the effects of hydrolysis and
etching on silica shells grown with different
kinetic rates.
•Investigating how the silica shell instability
impacts targeting of these nanoparticles.
•Investigating the effect of silica shell growth
kinetics on Si-O-Si bond formation.Synthesizing new Raman flavors and coatings for improved in vivo use
Dyes currently used for SERS imaging
IR-780 perchlorate BPE
CH 3 H 3C N
CH 3 H 3C
N N
N
H 3C CH 3
ClO4-
IR-792 perchlorate Areas of
improvement
• Non-fluorescent dyes
H 3C CH 3
H 3C
such as BPE not
resonant with 785 nm
H 3C
S
N
N
CH 3
laser.
H 3C • Resonant dyes such
ClO4-
as IR series have
high fluorescence
background
With Stefan Harmsen, InstructorResonant, low-fluorescent Raman dyes for better
multiplexing and sensitivity
N N HN
N
SiCl2: UV-Vis S01382: UV-Vis S0772: UV-Vis
2.0 20 30
1.5 15
20
Abs
Abs
Abs
1.0 10
10
0.5 5
0.0 0 0
400 600 800 400 600 800 400 600 800
Wavelength (nm) Wavelength (nm) Wavelength (nm)Attachment and silication of each Raman NP
SiCl2 S0772 IR-780 perchlorate
CH 3 H 3C
CH 3 H 3C
N N
N N
H 3C CH 3
ClO4-
Raman Spectra of SERS NP Normalized Raman Spectra
S0772 S0772
SiCl2 SiCl2
IR-780 IR-780
500 1000 1500 500 1000 1500
Raman Shift (cm-1) Raman Shift (cm-1)Upcoming aims for SERS NP 1. Determine limit of sensitivity of each SERS nanoparticle with optimized synthesis. 2. Explore multiplexing capabilities and add to current library of SERS flavors. 3. Applications in vivo including ex vivo cell labeling and targeting the SERS NP. 4. Replacing silica shell with carboxylmethyl dextran for improved biocompability.
Imaging Natural Killer Cells via the
NKp-46 receptorNatural Killer cells’ role in immunity
1. Killing of target cells is
induced when activating
receptor signals >>
inhibitory receptor signals.
1. Main mechanism:
perforin & granzyme-
mediated apoptosis
2. Also: death-receptor
mediated apoptosis (NK
express TNF-related
apoptosis-inducing ligand,
TRAIL, and FasL)
Vivier et al, Nat. Rev. Immunol. 12 : 239, 2012Choosing an NK cell target for imaging
Some Human NK Cell Activating Receptors
FcRg FcRg FcRg
CD3z CD3z DAP12 CD3z DAP12 DAP12 DAP12 DAP10
- ITSM
*
- ITAM
YxxM
NKp46 NKp30 NKp40 CD16 CD244 KIR2DS2 KIR2DS1 CD94:NKG2C NKG2D CD160
(NCR1) (NCR3) (NCR2) (FcgRIIIA) (2B4) KIR2DS3 /E (BY55)
KIR2DS4
KIR2DS5
KIR3DS1
NK Cell
Adapted from NK Cells: Receptors and Functions, Vivier & Ugolini, Nat. Rev. Immunol.
PosterFlow cytometry on splenocytes
Marker Description
CD3 T-cells
CD49B expressed on NK
NKp46
cells, a subset of
splenic CD4+ T
cells
PD1 inhibitory molecule
expressed by
activated B and T
cells
KLRG1 inhibitory,
expressed by
subsets of natural
killer cells (NKs)
and effector and
memory T cells
CD69 rapidly induced on
activated T and B
cells, neutrophils, NK1.1
and NK cells
NKp46 Activating
receptor NK cells
NK1.1 NK cells and NK-Results: 64Cu-NKp46 radiosynthesis
Specification Quality
O
Average DOTA molecules 4.5
per antibody
O
N
N
HO
OH
Radiochemical yield 70-75%
N
N
OH
O
O
Radiochemical purity >98%
(TLC)
Clone 29A1.4
Rat IgG2a, κ
Specific activity 5-10
µCi/µgResults: 64Cu-NKp46 stability
PBS Stability
100
Free 64Cu
80 NKp-46 (10 ug)
NKp-46 (20 ug)
% bound
60
IgG2A (10 ug)
40 IgG2A (20 ug)
20
0
ur
ur
ur
ho
ho
ho
0
24
48In vivo study design with B16 allografts
d -8 Inject 3.5 x 105
B16 cells into left flank
d0
d 1, 2
Conduct PET/CT Radiolabel DOTA-NKp-46
Euthanize at 48H (or IgG control) with 64Cu
Inject 150-200 !Ci (15-20
!g)First PET/CT images of NKp-46 in B16 allografts
NKp-46 48H, n=3 NKp-46 48H, n=1
15% Id/g 15% Id/g
1% Id/g 1% Id/gFuture Aims for NK cell imaging • Continue NKp46 target validation in splenocytes and tumors. • Investigate the affect therapies such as Bortezimib has on NK cell populations/receptors. • Repeat imaging study in B16 cells with blocking study, isotype controls. • Image NK cell therapies with NKp46. • Investigate activating receptors with upregulated expression in activated NK cells, such as NKp30 and NKp44 (human NK cells only). • SCID mouse imaging.
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