8/18/2011
Magnetic Nanoparticle
Biomarkers in Thermal
Therapy and Screening
John B. Weaver
Department of Radiology,
Dartmouth Medical School &
Dartmouth-Hitchcock Medical Center
Outline
Introduction to Iron Oxide
Nanoparticle Thermal Therapy.
Introduction to MSB.
Nanoparticle Delivery:
Bound State Populations
Ovarian Screening:
Finding Microscopic Disease
John Weaver has no conflicts of interests.
1. Normal
Vessels Deliver
Targeted
Nanoparticles
2. Nanoparticles
Uniformly
Distributed
3. Antibodies Bind
Nanoparticles to
Cancer Cells
Normal Tissue
Normal Cell
Cancer
Cancer Cell
with Targeted
Antigen
4. Treatment
(Superior AFM
Improved
Cytotoxicity)
1
8/18/2011
1. Normal
Vessels Deliver
Targeted
Nanoparticles
2. Nanoparticles
Uniformly
Distributed
3. Antibodies Bind
Nanoparticles to
Cancer Cells
Normal Tissue
Normal Cell
Cancer
Cancer Cell
with Targeted
Antigen
Advantages: Localization
Cell targeting with Nanoparticles
AMF penetration to heat deep tumors
Challenges:
Nanoparticle delivery (common to all NPs)
Lower SAR than optical heating
Heat localization
Therapy monitoring
4. Treatment
(Superior AFM
Improved
Cytotoxicity)
P Keblinskia, DG Cahill, A Bodapati, CR Sullivan, TA Taton,
“Limits of localized heating by electromagnetically excited nanoparticles”
Journal Of Applied Physics 100:054305, 2006.
Abstract:
Based on an analysis of the diffusive heat flow equation, we
determine limits on the localization of heating of soft materials and
biological tissues by electromagnetically excited nanoparticles.
For heating by rf magnetic fields or heating by typical continuous
wave lasers, the local temperature rise adjacent to magnetic or
metallic nanoparticles is negligible.
However, heat dissipation for a large number of nanoparticles
dispersed in a macroscopic region of a material or tissue produces
a global temperature rise that is orders of magnitude larger than the
temperature rise adjacent to a single nanoparticle.
One approach for producing a significant local temperature rise on
nanometer length scales is heating by high-power pulsed or
modulated lasers with low duty cycle.
Local heating of magnetic nanoparticles were used
to open and close cell membrane ion channels
MnFe2O4 nanoparticles (20 mg/ml, 6 nm) subjected to a RF magnetic field (40 MHz, 8.4 G) heated at
an initial rate of 0.62 oC/s.
H Huang, S Delikanli, H Zeng, DM Ferkey, A Pralle “Remote control of ion channels and neurons
through magnetic-field heating of nanoparticles” Nature Nanotechnology 5:602-6, 2010.
Maghemite anionic nanoparticles heated
(SLP 203 W g–1) at 700 kHz and 31 mT
JP Fortin, F Gazeau, C Wilhelm “Intracellular heating of living cells through Neel relaxation of magnetic
nanoparticles” Eur Biophys J 37:223–8, 2008.
Preferential thermal diffusion along molecules
rather than through surrounding liquids.
K Hamad-Schifferli, JJ Schwartz, AT Santos, S Zhang, JM Jacobson “Remote electronic control of
DNA hybridization through inductive coupling to an attachedmetal nanocrystal antenna” Nature
415:152-5, 2002.
2
8/18/2011
P. Jack Hoopes, Jen Tate, Jess Ogden
0.5
0.0
W41 - cells kept at 41 oC for
10 min using a waterbath.
10
20
30
Time After Treatment (hours)
0
37OC Water Bath
37OC AMF
Apoptotic
Dead
Not
Apoptotic
Alive
P41 - cells kept
at 41 oC for 10
min using
nanoparticle
heating.
1.0
Iron nanoparticles injected IP, ascites removed after 2 hours kept at
37OC for 30 minutes and then assayed (flow cytometry) 18 hours later.
PI Staining (PE)
2.0
1.5
Jay Baird, Steve Fiering Lab
Lack of intact membrane
Absorbance
(proportional to dead cells)
LDH (lactate dehydrogenase) measure of cell death:
a) microscopically localized nanoparticle heating and
b) non-localized heating
Annexin V - FITC
Antibody marking initiation of apoptosis
Greater cell death for nanoparticle heating reflects importance
of proximity of NPs to cells; i.e., the microscopic bound state.
Jay Baird, Steve Fiering Lab
AMF can kill cells containing iron nanoparticles without
global heating in vitro
4.5% of
Cells
Dead
or
Dying
PI Staining (PE)
37OC Water Bath
0.5%
1%
3%
Annexin V - FITC
Outline
37OC AMF
3%
13%
11%
27% of
Cells
Dead
or
Dying
Introduction to Iron Oxide
Nanoparticle Thermal Therapy.
Introduction to MSB.
Nanoparticle Delivery:
Bound State Populations
Ovarian Screening:
Finding Microscopic Disease
3
8/18/2011
MSB - Magnetic
Spectroscopy of Nanoparticle
Brownian Motion
MSB in vitro Apparatus
Input
Fourier transform of the H field
is a single peak because it is a
pure sinusoid
Flat top
Phase lag
Audio Power
Amplifier
Output
Fourier Transforms
H drive field
Sinusoidal
Signal
Generator
Resonant
Drive Coil
Pickup Coil
Nanoparticle
Sample
Magnetization
Fourier transform of the magnetization has
many harmonics because of the “squared
off” magnetization due to hysteresis.
For larger nanoparticles the rotational
Brownian motion determines:
the shape of the magnetization
measured by ratio of the harmonics.
the phase lag of the magnetization.
MSB in vivo Apparatus
Preamp &
Filter
Computer
Fast ADC
The harmonics are only produced
by magnetic nanoparticles.
Nanogram sensitivities in vivo.
MSB signal is affected by:
Temperature J.B. Weaver, A.M. Rauwerdink, E.W. Hansen, “Magnetic Nanoparticle Temperature
Estimation”,
Medical
Viscosity
- Physics 36(5):1822-1829 (2009).
A.M. Rauwerdink, J.B. Weaver, “Viscous effects on nanoparticle magnetization
A.M.
Rauwerdink,
Hansen, J.B.
“Nanoparticle
temperature
estimation
harmonics”
JournalE.W.
of Magnetism
andWeaver,
Magnetic
Materials 322:609-613
(2010).
in combined ac and dc magnetic fields”, Phys. Med. Biol. 54:L51–L55 (2009).
A.M. Rauwerdink, J.B. Weaver, “Measurement of Molecular Binding Using The
Brownian Motion of Magnetic Nanoparticle Probes” Applied Physics Letters 96,
033702 (2010).
J.B. Weaver, A.M. Rauwerdink, E.W. Hansen, “Magnetic Nanoparticle Temperature
Estimation”, Medical Physics 36(5):1822-1829 (2009).
Binding -
Can also measure (quantify) these effects.
A.M. Rauwerdink, E.W. Hansen, J.B. Weaver, “Nanoparticle temperature estimation
in combined ac and dc magnetic fields”, Phys. Med. Biol. 54:L51–L55 (2009).
J.B. Weaver, “MSB Relaxation Estimation”, in preparation.
4
8/18/2011
1. Normal
Vessels Deliver
Targeted
Nanoparticles
Outline
Introduction to Iron Oxide
Nanoparticle Thermal Therapy.
Introduction to MSB.
Nanoparticle Delivery:
Bound State Populations
Ovarian Screening:
Finding Microscopic Disease
1. Vascular
Compartment
Normal Tissue
Normal Cell
2. Porous Vessels
Allow Nanoparticles
into the Interstitial
Spaces
3. Antibodies Bind
Nanoparticles to
Cancer Cells
Normal Tissue
Normal Cell
Cancer
Cancer Cell
with Targeted
Antigen
4. Binding Mediated
Endocytosis
5. Superior AFM
Induced Cytotoxicity
Estimates of Average Binding
Debye approximation (low amplitude fields):
Fokker–Planck equation:
2. Interstitial Space
Cancer
3. Cell Surface
Bound
Cancer Cell
with Targeted
Antigen
4. Vesicles
5
8/18/2011
Calibration Sweep
40nm Iron Oxide Nanoparticles
Harmonic Ratio
Bound
Measurement
tb = εtf
where ε>1
ωLtf ω1tf
ω1tb ωHtf
ωt
Harmonic Ratio
Calibration Sweep
Bound
Measurement
ωL
ω1
εω1
ωH
ω
Methods:
- Bound State Concentrations
- MSB Measurement from Bound State 1
- MSB Measurement from Bound State 2
- MSB Measurement from the
Combination of Bound States
A.M. Rauwerdink, J.B. Weaver “Concurrent quantification of nanoparticle bound states”,
Medical Physics 38(3):1136-1140, 2011.
6
8/18/2011
Mixture of Two Bound States
Mixture of Two Bound States:
RMS Errors in the
Estimated
Concentrations:
Streptavidin
= 0.1023 mg
(0.4% of 25mg)
Bead
= 0.0994 mg
(0.4% of 25mg)
290 Hz (11mT)
One Measurement
290 Hz (11mT)
One Measurement
A.M. Rauwerdink, J.B. Weaver “Concurrent quantification of nanoparticle bound
states”, Medical Physics 38(3):1136-1140, 2011.
A.M. Rauwerdink, J.B. Weaver “Concurrent quantification of nanoparticle bound
states”, Medical Physics 38(3):1136-1140, 2011.
Mixture of Three Bound States:
RMS Errors in the
Concentration of:
Streptavidin bound
= 0.2770 mg
(1.1% of 25mg)
Bead bound
= 0.3177 mg
(1.3% of 25mg)
Glycerol bound
= 0.2152 mg
(0.86% of 25mg)
A.M. Rauwerdink, J.B. Weaver “Concurrent quantification of nanoparticle bound
states”, Medical Physics 38(3):1136-1140, 2011.
290 Hz (11mT & 16mT)
Two Measurements
Concentrations of well characterized
bound states measured with MSB:
RMS Errors in the concentrations of:
two microscopic bound states 0.4% error:
Streptavidin 0.1023 mg (0.4% of 25mg)
Bead 0.0994 mg (0.4% of 25mg)
three microscopic bound states 1.1% error:
Streptavidin bound 0.2770 mg (1.1% of 25mg)
Bead bound 0.3177 mg (1.3% of 25mg)
Glycerol bound 0.2152 mg (0.86% of 25mg)
A.M. Rauwerdink, J.B. Weaver “Concurrent quantification of nanoparticle bound states”, Medical Physics 38(3):11361140, 2011.
Quantification of different nanoparticle amounts.
A.M. Rauwerdink, A.J. Giustini, J.B. Weaver “Simultaneous quantification of multiple magnetic nanoparticles”,
Nanotechnology 21(45):455101 (2010).
7
8/18/2011
in vitro Cell Uptake
in vitro Cell Uptake
TEM images of nanoparticles in solution with MTG-B cells.
MSB Harmonics Over Time
3rd
(a) 5 minutes
(b) 5 hours
post addition of nanoparticles
Nanoparticles are (a) outside cells
(b) in vesicles and on cell surface.
290 Hz and 16 mT/μ0.
Adam M. Rauwerdink (& J.B. Weaver)
Andrew J. Giustini (& P.J. Hoopes)
in vitro Cell Uptake
5th Harmonic
Harmonic
Adam M. Rauwerdink (& J.B. Weaver)
Andrew J. Giustini (& P.J. Hoopes)
Cells Uptake Measurements
290 Hz
16 mT/μ0
Protein Binding Cell Surface and in Media
No Increased Binding
No Increased Binding
MCF-7 Uptake
less than
BT-474 Uptake 1
Invagination & Protein Binding
1
Steinhauser, I., Spankuch, B.,
Strebhardt, K., Langer, K.,
2006. Biomaterials 27, 49754983.
Cool Cells to Eliminate Invagination
Adam M. Rauwerdink (& J.B. Weaver)
Andrew J. Giustini (& P.J. Hoopes)
8
8/18/2011
in vivo Tumor Uptake Studies
Harmonic Ratio vs Incubation Time in Tissue
Number of
Nanoparticles
Average Metrics for Disperse
Binding Energies
1.
2.
3.
4.
5.
6.
Number of
Nanoparticles
Binding Energy
Direct Injection
Incubation
Tumor Excision
Tumor Division
MSB Measurement
Pathology
~20% error in calculated
bound states in simulations
Adam M. Rauwerdink (& J.B. Weaver)
Andrew J. Giustini (& P.J. Hoopes)
Binding Energy
Outline
MSB can estimate
bound state
populations with
good accuracy with
in vivo potential.
MSB has the
potential to describe
poorly characterized
bound states.
Introduction to Iron Oxide
Nanoparticle Thermal Therapy.
Introduction to MSB.
Nanoparticle Delivery:
Bound State Populations
Ovarian Screening:
Finding Microscopic Disease
9
8/18/2011
Early Detection with Iron Oxide NPs
Method: 24 hrs post ip NP inj. in mice
D21
D35
13mm3
D59
180mm3
P53/Kras Tg
WT
15,750mm3
20
13
11
Contralateral
34
24
13
AdvCre
14
13
13
Contralateral
14
12
13
AdvCre
Time Following NP Injection
MSB Signal
CLCL
AdvCre
Adv Cre
MSB signal from the contralateral and Ad-Cre side
of both transgenic (Tg) and wild type (WT) mice 24
days after Ad-cre injection (lesion still microscopic)
Magnetic detection
of nanoparticles
(MSB) used in
conjunction with
immunological
targeting has the
potential to identify
microscopic ovarian
cancer.
10
8/18/2011
Support:
Magnetic spectroscopy of nanoparticle
Brownian motion (MSB) can:
1) Estimate bound state populations with
good accuracy with in vivo potential.
2) Identify microscopic ovarian cancer
with immunological targeted
nanoparticles.
1) NIH - Center for Cancer
Nanotechnology Excellence
1U54CA151662-01
2) Norris Cotton Cancer Center
3) Department of Radiology
4) Thayer School of Engineering
Thank You!
11
8/18/2011
P Keblinskia, DG Cahill, A Bodapati, CR Sullivan, TA Taton,
“Limits of localized heating by electromagnetically excited nanoparticles”
Journal Of Applied Physics 100:054305, 2006.
Abstract:
Based on an analysis of the diffusive heat flow equation, we
determine limits on the localization of heating of soft materials and
biological tissues by electromagnetically excited nanoparticles.
For heating by rf magnetic fields or heating by typical continuous
wave lasers, the local temperature rise adjacent to magnetic or
metallic nanoparticles is negligible.
However, heat dissipation for a large number of nanoparticles
dispersed in a macroscopic region of a material or tissue produces
a global temperature rise that is orders of magnitude larger than the
temperature rise adjacent to a single nanoparticle.
One approach for producing a significant local temperature rise on
nanometer length scales is heating by high-power pulsed or
modulated lasers with low duty cycle.
Local heating of magnetic nanoparticles were used
to open and close cell membrane ion channels
MnFe2O4 nanoparticles (20 mg/ml, 6 nm) subjected to a RF magnetic field (40 MHz, 8.4 G) heated at
an initial rate of 0.62 oC/s.
H Huang, S Delikanli, H Zeng, DM Ferkey, A Pralle “Remote control of ion channels and neurons
through magnetic-field heating of nanoparticles” Nature Nanotechnology 5:602-6, 2010.
Maghemite anionic nanoparticles heated
(SLP 203 W g–1) at 700 kHz and 31 mT
JP Fortin, F Gazeau, C Wilhelm “Intracellular heating of living cells through Neel relaxation of magnetic
nanoparticles” Eur Biophys J 37:223–8, 2008.
Preferential thermal diffusion along molecules
rather than through surrounding liquids.
K Hamad-Schifferli, JJ Schwartz, AT Santos, S Zhang, JM Jacobson “Remote electronic control of
DNA hybridization through inductive coupling to an attachedmetal nanocrystal antenna” Nature
415:152-5, 2002.
P. Jack Hoopes, Jen Tate, Jess Ogden
0.5
0.0
W41 - cells kept at 41 oC for
10 min using a waterbath.
0
10
20
Time After Treatment (hours)
37OC Water Bath
37OC AMF
Apoptotic
Dead
Not
Apoptotic
Alive
1.0
Iron nanoparticles injected IP, ascites removed after 2 hours kept at
37OC for 30 minutes and then assayed (flow cytometry) 18 hours later.
PI Staining (PE)
P41 - cells kept at
41 oC for 10 min
using nanoparticle
heating.
1.5
Jay Baird, Steve Fiering Lab
Lack of intact membrane
Absorbance
(proportional to dead cells)
LDH (lactate dehydrogenase) measure of cell death:
a) microscopically localized nanoparticle heating
and
2.0
b) non-localized
heating
Annexin V - FITC
30
Antibody marking initiation of apoptosis
Greater cell death for nanoparticle heating reflects importance
of proximity of NPs to cells; i.e., the microscopic bound state.
12
8/18/2011
Jay Baird, Steve Fiering Lab
AMF can kill cells containing iron nanoparticles without
global heating in vitro
4.5% of
Cells
Dead
or
Dying
PI Staining (PE)
37OC Water Bath
0.5%
1%
37OC AMF
3%
3%
13%
11%
27% of
Cells
Dead
or
Dying
Adam Rauwerdink,
Thayer School of Engineering
Irina Perreard,
Department of Radiology
Andrew J. Giustini,
Thayer School of Engineering
P Jack Hoopes,
Department of Surgery
Annexin V - FITC
Killing is proportional to amount of heat
but this does not prove it is heat
Debye approximation:
Effective Field approximation to
the Fokker–Planck equation:
13
8/18/2011
Magnetic detection of nanoparticles (MSB):
1) Estimate bound state populations with good
accuracy with in vivo potential.
2) Estimate nanoparticle temperature.
3) Can be used in conjunction with immunological
targeting to identify microscopic ovarian cancer.
Outline
Introduction to Iron Oxide
Nanoparticle Thermal Therapy.
Introduction to MSB.
Nanoparticle Delivery:
Bound State Populations
Ovarian Screening:
Finding Microscopic Disease
John Weaver has no conflicts of interests.
Magnetic Nanoparticle
Biomarkers in Thermal Therapy
and Screening
John B. Weaver
Department of Radiology,
Dartmouth Medical School &
Dartmouth-Hitchcock Medical Center
14
8/18/2011
Harmonic Ratio vs Aggregate Size
D21
420 Hz, 22.9 mT/μ0
Gelatin bound
sample compares
closely with the
response of the
highly aggregated
nanoparticles.
13mm3
D35
180mm3
D59
15,750mm3
Adam M. Rauwerdink (& J.B. Weaver)
Andrew J. Giustini (& P.J. Hoopes)
15
8/18/2011
Linear correlation coefficient was:
0.991 for the 570Hz data and
0.994 for the 2100Hz data
Early Detection with Iron Oxide NPs
Time Following NP Injection
Method: 24 hrs post ip NP inj. in mice
CLCL
AdvCre
Adv Cre
WT
MSB Signal
P53/Kras Tg
20
13
11
MSB signal from the contralateral and Ad-Cre side
of both transgenic (Tg) and wild type (WT) mice 24
days after Ad-cre injection (lesion still microscopic)
Contralateral
34
24
13
AdvCre
14
13
13
Contralateral
14
12
13
AdvCre
16
8/18/2011
D21
13mm3
D35
180mm3
D59
15,750mm3
Outline
Delivery:
Bound State Populations
Temperature Monitoring:
Monitoring Thermal Therapy
Ovarian Screening:
Finding Microscopic Disease
17
8/18/2011
P Keblinskia, DG Cahill, A Bodapati, CR Sullivan, TA Taton,
“Limits of localized heating by electromagnetically excited nanoparticles”
Journal Of Applied Physics 100:054305, 2006.
Abstract:
Based on an analysis of the diffusive heat flow equation, we
determine limits on the localization of heating of soft materials and
biological tissues by electromagnetically excited nanoparticles.
For heating by rf magnetic fields or heating by typical continuous
wave lasers, the local temperature rise adjacent to magnetic or
metallic nanoparticles is negligible.
However, heat dissipation for a large number of nanoparticles
dispersed in a macroscopic region of a material or tissue produces
a global temperature rise that is orders of magnitude larger than the
temperature rise adjacent to a single nanoparticle.
One approach for producing a significant local temperature rise on
nanometer length scales is heating by high-power pulsed or
modulated lasers with low duty cycle.
Local heating of magnetic nanoparticles were used
to open and close cell membrane ion channels
MnFe2O4 nanoparticles (20 mg/ml, 6 nm) subjected to a RF magnetic field (40 MHz, 8.4 G) heated at
an initial rate of 0.62 oC/s.
H Huang, S Delikanli, H Zeng, DM Ferkey, A Pralle “Remote control of ion channels and neurons
through magnetic-field heating of nanoparticles” Nature Nanotechnology 5:602-6, 2010.
Maghemite anionic nanoparticles heated
(SLP 203 W g–1) at 700 kHz and 31 mT
JP Fortin, F Gazeau, C Wilhelm “Intracellular heating of living cells through Neel relaxation of magnetic
nanoparticles” Eur Biophys J 37:223–8, 2008.
Preferential thermal diffusion along molecules
rather than through surrounding liquids.
K Hamad-Schifferli, JJ Schwartz, AT Santos, S Zhang, JM Jacobson “Remote electronic control of
DNA hybridization through inductive coupling to an attachedmetal nanocrystal antenna” Nature
415:152-5, 2002.
P. Jack Hoopes, Jen Tate, Jess Ogden
Absorbance
(proportional to dead cells)
LDH (lactate dehydrogenase) measure of cell death:
a) microscopically localized nanoparticle heating
and
2.0
b) non-localized
heating
Immature DCs are
strongly phagocytic and
take up nanoparticles
aggressively
P41 - cells kept at
41 oC for 10 min
using nanoparticle
heating.
1.5
1.0
0.5
0.0
Jay Baird, Steve Fiering Lab
Iron nanoparticles injected IP, ascites removed after 2 hours kept at
37OC for 30 minutes and then assayed (flow cytometry) 18 hours later.
W41 - cells kept at 41 oC for
10 min using a waterbath.
0
10
20
Time After Treatment (hours)
30
Greater cell death for nanoparticle heating reflects importance
of proximity of NPs to cells; i.e., the microscopic bound state.
Iron nanoparticles injected IP into late stage ovarian
cancer mouse with ascites (60% tumor cells 40% VLC).
Iron stains with prussian blue.
95% of uptake is by phagocytes.
Nanoparticles dextran coated, no specific targeting.
18
8/18/2011
Jay Baird, Steve Fiering Lab
Jay Baird, Steve Fiering Lab
AMF can kill cells containing iron nanoparticles without
global heating in vitro
Iron nanoparticles injected IP, ascites removed after 2 hours kept at
37OC for 30 minutes and then assayed (flow cytometry) 18 hours later.
37OC Water Bath
Apoptotic
Dead
Not
Apoptotic
4.5% of
Cells
Dead
or
Dying
PI Staining (PE)
37OC AMF
Alive
PI Staining (PE)
Lack of intact membrane
37OC Water Bath
1%
0.5%
27% of
Cells
Dead
or
Dying
Annexin V - FITC
Killing is proportional to amount of heat
but this does not prove it is heat
Temperature Measurement:
A
B
C
Harmonics
T and H appear only as a ratio so M is
identical for
11%
Harmonics
μ – Nanoparticle Bulk Magnetization
Ho – magnitude of the drive field
k – Boltzmann Constant
T - Temperature
13%
Harmonics
Langevin function describes nanoparticle
magnetization in equilibrium:
3%
3%
Annexin V - FITC
Antibody marking initiation of apoptosis
Noninvasive Temperature Measurement:
37OC AMF
Hi/To
H/T
Change drive amplitude,
Hi, at constant
temperature, To, to find
the calibration curve
Ho/T1 H/T
First measurement at a
selected drive field, Ho,
yields a value of Ho/T1
from which the current
temperature, T1, can be
found
Ho/T2
H/T
Subsequent
measurements at the
selected drive field yield
values of Ho/T2 from which
the current temperature,
T2, can be found
19
8/18/2011
Temperature Estimation Accuracy:
Magnetic detection of
nanoparticles (MSB) can
estimate nanoparticle
temperature.
MSB has the potential to
do so during therapy.
20
8/18/2011
Explore the
correlation between:
a) microscopic
collagen stiffness
and
b) macroscopic
tissue stiffness.
MSB micro-rheology
Explore
microscopic
stiffness in vivo.
1) Tumor stroma has
increased fibrotic
foci.
2) Increased levels of
the crosslinking
(lysyl oxidase).
3) Increase in matrix
metalloproteinases
that disgest the
extracellular matrix
allowing cells to
migrate &
angiogenesis.
MSB micro-rheology
21
8/18/2011
Number of
Nanoparticles
Average Metrics for Disperse
Binding Energies
Estimates of Average Binding
Debye approximation (low amplitude fields):
Fokker–Planck equation:
Number of
Nanoparticles
Binding Energy
Binding Energy
22
8/18/2011
Calibration Sweep
Estimates of Average Binding
Harmonic Ratio
Debye approximation (low amplitude fields):
Bound
Measurement
tb = εtf
where ε>1
Effective Field approximation to
the Fokker–Planck equation:
ωLtf ω1tf
ω1tb ωHtf
ωt
Harmonic Ratio
Calibration Sweep
Bound
Measurement
ωL
ω1
εω1
ωH
ω
40nm Iron Oxide Nanoparticles
23
8/18/2011
24
8/18/2011
If the harmonics depend on the product ν, then decreasing  by
a factor ε and increasing ν by the same factor produces the
same harmonic because the τν product remains the same.
A
Harmonic Ratio
MSB Estimates of Bound Fraction:
nH  B
Calibration Sweep, τc
nL  c
nL  B
nH  c
Bound Sweep, B
Harmonic Ratio
c <
B
ν/ε
ε caused by binding
(ν/ε) (ε) = 1
Frequency
Bound Sweep, B
Calibration Sweep, τc
nL
The long-term goal of Project 2 is to develop
methodology to quantify nanoparticle binding
and microscopic bio-distribution in vivo.
Combine two methods of measuring
binding in vivo:
Magnetic spectroscopy of nanoparticle
Brownian motion (MSB)
MSB measures the nanoparticle rotational
motion to estimate binding.
Ratiometric fluorescence spectroscopy (RFS)
RFS measures differential residence time
from targeted and untargeted agents.
n
Harmonic Ratio
B
nH
n
Differentiate enhanced permeability and
retention (EPR)1 from antibody binding.
4. Vesicle
Compartment
2. Interstitial Fluid
Compartment
1. Vascular
Compartment
3. Cell Surface
Bound Compartment
Quantify the distribution of nanoparticles
within four microscopic compartments.
[1] AI Minchinton, IF Tannock. “Drug penetration in tumors” Nature Reviews Cancer 6:583-592 (2006).
25
8/18/2011
MSB Estimates of Bound Fraction:
Two Bound States
If the binding
energies are
known for both
bound states, the
bound fraction can
be measured using
a single MSB
measurement.
Measured harmonics of
the unbound and bound
nanoparticles alone
- Bound State Concentrations
- MSB Measurement from Bound State 1
- MSB Measurement from Bound State 2
- MSB Measurement from the
Combination of Bound States
Methods:
Mixture Model:
Calculated
fractions for each
bound state
Methods:
The MSB signal from each of the tubes was
measured. The signal for each bound state
was estimated as the average of the signals
from each tube.
Measured
harmonics of
the mixture
1) 5 tubes with 5 mg iron targeted magnetic
nanoparticles in solution (Streptavidin bound),
were made.
2) 5 tubes with 5 mg iron targeted magnetic
nanoparticles bound to 2mm beads, were made.
3) 4 tubes with 5 mg iron targeted magnetic
nanoparticles (Streptavidin bound) in
glycerol/water solution (2cP).
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8/18/2011
Mixture of Two Bound States
Mixture of Two Bound States:
RMS Errors in the
Estimated
Concentrations:
Streptavidin
= 0.1023 mg
(0.4% of 25mg)
Bead
= 0.0994 mg
(0.4% of 25mg)
290 Hz (11mT)
One Measurement
290 Hz (11mT)
One Measurement
Mixture of Three Bound States:
RMS Errors in the
Concentration of:
Streptavidin bound
= 0.2770 mg
(1.1% of 25mg)
Bead bound
= 0.3177 mg
(1.3% of 25mg)
Glycerol bound
= 0.2152 mg
(0.86% of 25mg)
290 Hz (11mT & 16mT)
Two Measurements
MSB measures of well characterized
bound states in vitro:
RMS Errors in the concentrations of:
two microscopic bound states 0.4% error:
Streptavidin 0.1023 mg (0.4% of 25mg)
Bead 0.0994 mg (0.4% of 25mg)
three microscopic bound states 1.1% error:
Streptavidin bound 0.2770 mg (1.1% of 25mg)
Bead bound 0.3177 mg (1.3% of 25mg)
Glycerol bound 0.2152 mg (0.86% of 25mg)
We are characterizing the in vivo bound
states to duplicate these results in animals.
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8/18/2011
Adam Rauwerdink,
Thayer School of Engineering
Irina Perreard,
Department of Radiology
Andrew J. Giustini,
Thayer School of Engineering
P Jack Hoopes,
Department of Surgery
Support:
1) NIH - Center for Cancer
Nanotechnology Excellence
1U54CA151662-01
2) Norris Cotton Cancer Center
3) Department of Radiology
4) Thayer School of Engineering
Thank You!
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8/18/2011
Debye approximation:
Thank You!
Effective Field approximation to
the Fokker–Planck equation:
NIH through NCCC Nanotechnology Seed Projects
Department of Radiology, Dartmouth-Hitchcock Medical Center
Dartmouth Medical School, Dartmouth College
Thayer School of Engineering, Dartmouth College
RFS Images of Binding Rate In Vivo
(using 2-probe injection, 1 targeted & 1 untargeted to EGF receptor)
EGF receptor Targeted dye
AsPC1 tumor in pancreas
Control dye
AsPC1 tumor in pancreas
Pogue et al, J. Biomed Opt Letters, (2010)
29