Calcium Phosphate Nanocolloids
f Bioimaging
for
Bi i
i and
d Drug
D
Delivery
D li
James H. Adair
with
E.I. Altinoglu, B. M. Barth, H.S. Muddana, T.T. Morgan, T.J. Russin,
M.R. Parette*,, J. Kaiser,, T. Tabouillot,, A. Tabakovic,, C. McGovern,, S.
Shanmugavelandy, P.C. Eklund, J.K. Yun, Y. Heakal, A. Sharma, P.J.
Butler, G.P. Robertson, V. Ruiz-Velasco, J. Smith and M. Kester
Materials Science & Engineering, Bioengineering, Physics,
Anesthesiology and Surgery, and Pharmacology,
Penn State University, University Park and Hershey, PA
*K
Keystone
t
N
Nano, B
Boalsburg,
l b
PA
Ceramic and Composite Materials Center
An NSF Industry/University Cooperative Research Center
Cancer Nanotechnology, Going Small for Big Advances- Using Nanotechnology to Advance Cancer Diagnosis, Prevention 1
& Treatment, NIH Publication No. 04-5489 (2004).
Overview
™ Background: Calcium Phosphate
™ Calcium Phosphate Nanocomposite
Particle (CPNP)
in vitro and in vivo Bioimaging and Drug
•in
Delivery – Breast and Pancreatic Cancers
™ What is Photodynamic Therapy?
™ What is Deep Tissue PDT?
™Photodynamic Therapy of Human Breast
Cancer and Leukemia
™ Summary and Conclusions
2
Features of a Useful Nanoparticle
Bi i
Bioimaging
i and
dD
Drug D
Delivery
li
™ Inherently
I h
tl non-toxic
t i materials
t i l and
d
degradation products
™ Biologically
Bi l i ll or extrinsically
t i i ll controlled
t ll d
release of therapeutic agents
™ Small
S ll particle
ti l di
diameter:
t 20
20-200nm
200
™ Colloidally stable in physiological
conditions
diti
(pH,
( H ionic
i i strength,
t
th
macromolecular interactions, and
t
temperature
t
™ Can be targeted to cell/tissue of choice
3
Calcium Phosphate Nanocomposite Particle O
Overview
i
Calcium phosphosilicate matrix material
20 nm
Imaging agents and or drugs contained in matrix
ICG CPNPs
ICG‐CPNPs
PEG, Citrate, Avidin, Anti‐Bodies (e.g., anti‐ on exterior permit targeting
4
Calcium Phosphosilicate Nanocomposite Particle (CPNPs): A Broad Platform for Seeking, Treating and Tracking Human Disease
J.H. Adair, P.J. Butler, P.C. Eklund, M. Kester, J. Smith and collaborators
,
,
,
,
Materials Science & Engr., Bioengineering, Physics, Pharamacology, Surgery
Features
¾ Calcium phosphosilicate matrix material: bioresorbable, dissolution on demand within cells, hiigh particle number and encapsulated active agent concentrations
¾ Imaging agents and or drugs contained in non‐porous matrix
¾ Colloidally stable for extended times in physiological fluids: phosphate buffered saline, cell culture media blood
culture media, blood ¾ Citrate, PEG, Avidin, Anti‐Bodies (e.g., anti‐CD 71), other molecules (e g Gastrin epitopes) bioconjugated
(e.g., Gastrin epitopes) bioconjugated on surface permitting targeted delivery
20 nm
ICG‐CPNPs
ICG
CPNPs
Research Opportunities
¾ Bioimaging/bioassays
Bi i
i /bi
b th
both in vitro and in vivo of healthy and transformed cells and tissues
¾ Targeted or untargeted (i.e., EPR) delivery of chemo‐
therapeutics to cancer (breast
therapeutics to cancer (breast, pancreatic cancer, leukemia) ¾ Potentially, long term monitoring for cancer in general patient populuion
¾ Deep tissue (>6cm) photo
Deep tissue (>6cm) photo‐
dynamic therapy of cancer with novel ICG‐CPNPs
Coming Soon
Fluorescent NanoJackets
Fluorescent NanoJackets
Dye
λex/λem
Surface Functionalization
Cascade Blue
COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, 400/425 Avidin
Fluorescein
COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, (
),
,
,
,
475/515 Avidin
Red (Rh WT)
Red (Rh WT)
COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, 530/555 Avidin
Indocyanine Green
COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, 760/875 Avidin
J.H. Adair and M. Kester are CSO and CMO for Keystone Nano, respectively
6
Resorbable Calcium Phosphate - NanoComposite Particles
ACP
Tailorable Solubility
→ Time Release
Time Release
Tumor
de Groot et al., Chemistry of Calcium Phosphate Bioceramics, 1980
7
In‐‐Vitro In
Vitro and and in
in‐‐Vivo Drug Delivery and Fluorescent Imaging
Calcium Phosphate Nanocomposite Colloids
8
H. Muddana, T. Tabouillot, T.M. Morgan, J.H. Adair, P.J. Butler, NanoLetters, 2008
Melanoma Cell Imaging and Apoptosis
(a)
DAPI Fluorescent
50 μm
Rh WT CPNPs
Merged Rh WT CPNPs with DAPI Staining
(b)
DAPI Fluorescent
50 μm
Rh WT CPNPs
Merged Rh WT CPNPs with Cer10
and DAPI Staining
In vitro effect of Cer10‐CPNPs on melanoma cell survival and viability. Representative photomicrographs of cultured
melanoma UACC 903 cells exposed to Rh WT‐CPNPs without (a) and with (b) Cer10. Fluorescent Cer10‐CPNPs, unlike
control CPNPs, induce melanoma cell death. (c) MTS cytotoxicity assay demonstrating dosage responsive cytotoxic
actions of Cer10‐CPNPs.
Cer10 CPNPs Control CPNPs exhibit modest cytotoxicity at the highest particle number concentration.
concentration Values
are mean ± SEM for three independent experiments, each experiment replicated in triplicate.
M. Kester and G.P. Robertson et al., NanoLetters, 2008.
9
Light propagation
in biological tissue
• “Therapeutic Window” – Minimum in absorption
• Highly diffuse scatterer!
• How does light propagate
How does light propagate
in a dense scattering medium? medium?
• Radiative Transport Equation
– Diffusion Approximation
Diffusion Approximation
10
T. J. Russin et al., in preparation.
CPNPs: BioPhotonics
Eklund Russin Altinoglu Adair
Eklund, Russin, Altinoglu, Adair
• Photodynamic Therapy
hν
ICG (S
(T01 state)
Oxygen (T
(S10 state)
– Need model for depth limits
N d
d l f d th li it
• Fluorescent imaging
– Targeting
Targeting to breast tissue to breast tissue
achieved in nude mice
Altinoglu et al., ACS Nano, 2008
–N
Need a model to predict d
d lt
di t
imaging capabilities
11
Dissection 10 minutes post‐injection
ICG‐CPNP‐PEG Sample
Images show exclusive hepatic clearance of PEGylated CPNPs No significant sequestering or uptake by other tissues or membranes 12
13
B.M. Barth, C. McGovern,
R. Sharma, E.I. Altinoglu,
J.H. Adair, M. Kester, J.
Smith
14
What is Photodynamic Therapy?
• Photodynamic therapy (PDT) is a non‐invasive cancer treatment option with minimal side effects
• Involves activation of a non‐
toxic photosensitizer by an appropriate light source in the presence of molecular oxygen
f
l l
http://www.easternsuburbsderm.com.au/pdt.htm
Current Limitations
•Penetration Depth: ~50μm
with red light
•Systemic Patient PhotoSensitivity
15
http://sterileeye.com/
What is PDT?
What is PDT?
5
3
S12
1
0
No
on-radiative de
ecay
5
3
S02
1
0
Fluorescence
e
Vibrationa
l
relaxation
s
Absorption
Energy
• Upon irradiation (absorption p
(
p
of photons), the photosensitizer is excited to a higher energy state and
a higher energy state and upon return to its ground state, transfers this energy to oxygen
Intersyste
5m
3
T21crossing
1
0
(Singlet Oxygen)
Energy transfer
(Molecular Oxygen)
Photosensitizer (ICG)
Oxygen
• Both unstable radicals and highly reactive singlet oxygen p
,
,
are produced, which cause localized, lethal cellular damage in milliseconds
16
Photosensitizer: Indocyanine Green (ICG)
Photosensitizer: Indocyanine Green (ICG)
• Near IR emitting fluorophore
– Excitation: 785 nm Emission: 820 nm
Excitation: 785 nm Emission: 820 nm
• FDA approved, non‐toxic
• First proposed for PDT by Fickweiler First proposed for PDT by Fickweiler
et al. (1997)
– Showed
Showed ICG more effective than ICG more effective than
Photofrin
–
Fickweiler et al., Indocyanine green: Intracellular uptake and phototherapeutic effects in vitro, J Photochem Photobiol B 38 (1997) 178‐183
• NIR allows deeper penetration depths
– Biological transmission window in NIR
Biological transmission window in NIR
Fig.: Hamblin MR, Demidova TN. In:
Mechanisms for low
low-light
light therapy.
therapy Hamblin,
Hamblin
MR, Waynant, RW, Anders, J (Eds.) (SPIE,
Bellingham, WA, 2006) 614001-614013
17
Experimental Layout for
Penetration Depth & Bioimaging
h
Porcine tissue in
hanging clamp
Photodiode
power meter
25°
785 nm
laser diode
Infrared Camera
785 nm laser
on variable
angle mount
20 cm
Emitted light
solid angle Ω
53.6 mm
Optical table
Porcine tissue
Optical table
CPNP sample in
50 μL well
18
T. J. Russin et al., in preparation.
Penetration Depth for Photodynamic Therapy
h d
h
• Indocyanine Green PDT
– Radiation dose of 48 J/cm2
– μeff (pork)= 1.44
– μeff (breast)= 0.739
S. Fickweiler et al., J. Photochem. Photobiol. BBiol., 38 (1997).
19
T. J. Russin et al., in preparation.
Deep Tissue Photodynamic Therapy Deep
Tissue Photodynamic Therapy
(DTPDT)
with ICG‐CPNPs
B. Barth, S. Shanmugavelandy, E.I. Altinoglu, S. Knupp, J.H. Adair, l
l
d
M. Kester 20
Initial Animal Trials
Initial Animal Trials
• MDA‐MB‐231 human breast adenocarcinoma
3 u a b east ade oca c o a
• Nude mouse model; 5 groups (n=4)
1. ICG-CPNP-PEG
1
2. ICG-CPNP-COOH
3. Ghost-CPNP-PEG
4. Free ICG Control
4
5. PBS Blank Control
• 100 uL
100 uL tail vein injection
tail vein injection
– 2.5e‐7 M ICG absorption value
• Single
Single ~0.002
0.002 and 50 J/cm
and 50 J/cm2 (3 minutes) (3 minutes)
irradiation 24 hrs post injection
– 70 mW, 785 nm laser diode
21
Initial In Vivo Photodynamic Therapy of Breast Cancer Tumors ~0.002 J/cm2
ICG-CPNP Photodynamic Therapy
MDA-MB-231 Breast Cancer Xenografts
g
in Nude Mouse Model
3000
Rela
ative Tumor Size
2500
Ghost
2000
COOH
PEG
1500
1000
500
0
0
5
10
15
20
25
30
35
40
45
Days
•
Kodak In Vivo FX Imager used to deliver light to tumors
•
About 1/25000
About
1/25000th of reported photodynamic therapy light doses
of reported photodynamic therapy light doses
•
Light given 24 hours post‐injection
22
Second Trial In Vivo Photodynamic Therapy of 50
/ 2
Breast Cancer Tumors 50 J/cm
45
40
PBS
Relative Tumo
R
or Size
35
Ghost-CPNP-PEG
Free-ICG
30
ICG CPNP COOH
ICG-CPNP-COOH
25
ICG-CPNP-PEG
20
15
10
5
0
0
5
10
15
20
25
30
35
40
45
50
55
Days
23
Non Solid Tumors ‐ Leukemia
PDT Targeted to Spleen
PDT of KG-1 Xenografted Nude Mice
70
Ghost CPNP PEG
Ghost-CPNP-PEG
X1
1,000 WBC per ul
60
ICG-CPNP-PEG
50
C6-CPNP-PEG
40
ICG- + C6-CPNP-PEG
30
20
10
0
0
5
10
15
Days
20
25
24
Summary
‰ CPNPs encapsulate imaging agents, therapeutics or both, permitting both in vitro and in vivo simultaneous detecting, delivery, and monitoring for cellular uptake
delivery, and monitoring for cellular uptake
‰ Drug delivery of both hydrophobic and hydrophilic drugs is improved by the intercellular protection afforded by the calcium phosphate during transport to the cell combined with the on‐
demand dissolution of the CP and intracellular drug delivery
‰ Enhanced permeability and retention (EPR) in solid human Enhanced permeability and retention (EPR) in solid human
breast cancer tumors and pancreatic tumors is observed in vivo for a pegylated and targeted near infra‐red – CPNP with the promise of deep tissue imaging of lesions
i
fd
ti
i
i
fl i
‰ Photodynamic therapy with deep soft tissue penetration holds p
much promise in the treatment of cancer 25
The End
Thank You for Your Patience
Questions?
C
Courtesy
t
C
C. Si
Siedlecki
dl ki
26