Vascular Zip Codes in Nanoparticle Targeting
Erkki Ruoslahti
Burnham Institute at UCSB
and
Department of Molecular Cellular and Developmental Biology, UCSB
Ruoslahti lab
Santa Barbara
Hector Biliran
Priya Karmali
V.Ramana Kotamraju
David Peters
Barry Rowan
La Jolla
Austin Derfus
Valentina Fogal
Tero Jarvinen
Jan Pilch
Dmitri Simberg
Kazuki Sugahara
Tambet Teesalu
Miriam Wankell
Lianglin Zhang
Former lab
members
Maria Akerman
Sven Christian
Markus Essler
Pirjo Laakkonen
Kimmo Porkka
Support
NCI, NHLBI, DoD, Komen
Collaborators
Doug Hanahan, UCSF
Enrico Giraudo
Sangeeta Bhatia, MIT, UCSD,
Austin Derfus,
Todd Harris
Geoffrey von Maltzahn
Michael Sailor, UCSD
Ji HO Park
Michael Schwartz
Robert Hoffman, UCSD
Matthew Tirrell, UCSB
Patrick Daugherty, UCSB
Targeting Nanoparticles into Tumors
Modular Approach to Multifunctional Nanoparticles
Physically target:
- vascular cells
- tumor cells
- extracellular matrix
Trigger/engineer:
- tissue penetration
- RES avoidance
- subcellular targeting
- diagnostic signal(s)
- release of therapeutic
agent
- amplification of targeting
Ruoslahti, Cancer Cell, 2002
Isolation of homing peptides by ex vivo/in vivo phage screening
Ex vivo
Incubate
phage with
cells from
target tissue
Rescue phage
and amplify
Wash
Inject (enriched)
phage library
into a mouse iv
In vivo
Rescue and
amplify phage
from target
tissue
Phage bind to blood
vessels in target tissue
Repeat in vivo screening,
sequence individual phage
from enriched pool
Homing peptides
The blood vessels in individual tissues are distinct
350
Relative phage titer
Normal tissues targeted with
vascular homing peptides:
Heart-homing phage
300
250
Brain
Kidneys
Lungs
Prostate
Skin
Breast
Muscle
Pancreas
Adrenals
Uterus
Liver
Heart
200
150
100
50
0
Heart
Kidney
Brain
Muscle
Lung
- Bacterial 2-hybrid screen for receptors
L. Zhang et al. Circulation, 2005
Metastasis
Lymphangiogenesis
Angiogenesis
(blood vessels)
(lymphatics)
Tumor-homing Peptides
Peptide
Sequence
Specificity
Receptor
Blood Vessels
RGD-4C
CDCRGDCFC
Angiogenic endothelium
αvβ3 and αvβ3
integrins
CAR
Nonapeptide
Angiogenic endothelium
F3
34-amino acid
basic peptide
Angiogenic endothelium, Cell surface
tumor cells
nucleolin
Porkka et al.,
2002; Christian
et al., 2003
CGKRK
CGKRK
Angiogenic endothelium, tumor cells
Heparan
sulfate?
Hoffman et al.,
2003
Breast ca lymphatics,
tumor cells
Not known
Laakkonen et
al., 2002; 2004
LSD
Melanoma lymphatics
CXCR4?
REA
Prostate ca lymphatics,
tumor cells
Zhang et al,
submitted
Zhang et al,
submitted
Not known
Essler et al.,
unpublished
Lymphatic Vessels
LyP-1
CGNKRTRGC
Vessel/Tumor ECM
CREKA
CREKA
Plasma Clot
CLTI/2
10-amino acid
cyclic peptides
Breast ca extracellular
matrix
Blood clots, fibrin
deposits in tumors
Fibro
nectin
Reference
Arap et al.,
Science, 1998
Jarvinen et al.
Pilch et al.,
l.,
PNAS, 2006
Tumor Blood Vessels
F3 is a cell-penetrating peptide specific for tumor endothelial cells and tumor cells
FITC
F3 PEPTIDE
Inject i.v.
F3 i.v.- green
Blood vessels - red
Nuclei - blue
F3, HL-60 tumor
Add to tumor cell culture
F3
Anti-nucleolin i.v.- red
Blood vessels - green
F3 + antinucleolin
Control IgG i.v.- red
Blood vessels - green
Tumor-homing peptide CAR recognizes tumor vessels
and penetrates into tumor parenchyma
Blood vessels
FITC-CAR peptide
Nuclei
Merge
Jarvinen et al.
Fluorescein-labeled vascular homing peptides from in vivo phage display reveal
blood vessel changes that parallel progression in tumor development
Intravenously
injected peptide
Normal islet
Angiogenic islet
Tumor
1
2
3
Joyce et al., Cancer Cell, 2003; Hoffman et al., Cancer Cell, 2003
Tumor Lymphatics
LyP-1 peptide co-localizes with tumor lymphatics
FITC-LyP-1
Podoplanin
FITC-LyP-1
Lyve-1
Laakkonen et al.,Nature Med., 2002
20
IB
R
H
PV
sk
in
K
ca
nc
er
C
1
PP
tu
m
M
D
A
-M
B
C
81
61
B-
C8
16
1
0
0
or
10
m
p
20
30
Tr
a
40
40
tu
m
or
60
50
Py
M
T
80
LSD
-4
35
100
Fold over nonrecombinant phage
LyP-1
M
43 DA
5 -M
Fold over nonrecombinant phage
LyP-1 and LSD phage have mutually exclusive tumor type
specificities
Anti-podoplanin
Anti-LYVE-1
Anti-CD31
Lymphatic homing peptides for Breast cancer
Prostate cancer
Melanoma
Zhang et al., Cancer Res., 2006
Selective tissue damage by homing peptide-targeted
pro-apoptotic peptide D(KLAKLAK)2
RGD-4C = cCDCRGDCFC
Receptor: integrins αvβ3 and αvβ5
Ellerby et al. Nat. Med., 1999
Target: angiogenesis
Arap et al.
PNAS, 2002
Gerlag et al. Arthr. Res., 2001
Inhibition of C8161 tumor growth by D(KLAKLAK)2-LSD conjugate
1600
1400
D(KLAKLAK)2-LSD
D(KLAKLAK)2
1200
plus LSD
1000
800
600
**
400
200
0
16
20
24
28
32
36
Vessels per field
PBS
40
35
30
25
20
15
10
5
0
D(KLAKLAK)2-LSD
D(KLAKLAK)2 plus LSD
PBS
*
**
Blood vessels Lymphatic vesse
days post implantatio
L. Zhang et al.
Tumor imaging with a homing peptide for tumor lymphatics
140
Control peptide
120
LyP-1
LyP-1b
Fluorescence
100
80
60
40
20
0
T
B
H
Lu
Li
S
K
Laakkonen et al., PNAS, 2004
Extracellular Matrix
A Homing Peptide Binds to Clotted Plasma Proteins in a Tumor
A
B
C
tumor
liver
spleen
lung
brain
CLT1
D
E
F
Control
peptide
CLT1/
Normal
tissues
heart
spleen
lung
tumor heart
kidneybrain
G
H
I
J
K
L
liver
Pilch et al., PNAS, 2006
Nanomedicine
ZnS
CdSe
e
PEG
Pe
pt
id
L
Peptide
G
PE
e
PEG
T
Pe
pt
id
Pe
pt
id
e
PEG
Specific in vivo homing of quantum dots
F3-qdots
Tumor blood vessels
F3 (34 aa peptide)
LyP-qdots
Tumor blood vessels
cCGNKRTRGC
Lung-homing
qdots
cCGFECVRQCPERC
Akerman et al. PNAS, 2002
FI
TC
Peptide-coated magnetic particle
ID
PE
P
PE
PT
TI
DE
FI
TC
E
Dextran
Iron oxide
DE
TI
PE
PT
PEPTIDE
P
PE
ID
E
PEPTIDE
TC
FI
FI
TC
FITC
FITC
Proteolytically actuated self-assembly
Harris, von Maltzahn et al. (Bhatia laboratory) Angew. Chem., 2006
Role of PEG length and characterization of assembly
Harris, von Maltzahn et al. Angew. Chem., 2006
Triggered self-assembly of nanoparticles by tumor cells
HT-1080
HT-1080 +
inhibitor
HT-1080
HT-1080 + Inhibitor
Harris, von Maltzahn et al. Angew. Chem., 2006
Amplification of in vivo homing
Tissue distribution CREKA-coated iron oxide particles vs CREKA peptide
lymph nodes
tumor
liver
tumor
bladder
bladder
CREKA-IO
CREKA peptide
D. Simberg, T. Duza et al. (Ruoslahti laboratory)
Treatment of mice with decoy Ni-liposomes enhances tumor
homing by CREKA peptide-targeted IO nanoparticles
D. Simberg, T. Duza et al.
Ni-liposomes prolong blood half life of CREKA-IO nanoparticles
D. Simberg, T. Duza et al.
CREKA-IO accumulation in tumor vessels
after decoy particle treatment
A
x200
D. Simberg, T. Duza et al.
CREKA-IO nanoparticles accumulate
in a fibrin meshwork in vivo and in vitro
Tumor
Tumor
Fibrin
CREKA-IO
DAPI
CREKA-IO
Plasma clot in vitro
DAPI
CREKA-IO
- Inhibited by blood clotting inhibitor (heparin, hirudin)
D. Simberg, T. Duza et al.
Intravital imaging confirms clot formation in tumor vasculature in situ
20x
Vessel wall
D. Simberg, T. Duza et al.
Tumor imaging with decoy liposome-enhanced targeting
of CREKA-IO nanoparticles
D.Simberg et al.
Amplification of tumor targeting:
CREKA-IO particles induce homing of non-targeted Cy7-IO
particles
A
B
C
D.Simberg et al.
CREKA-IO particles may induce necrosis in tumor tissue
D. Simberg, et al.
• Blood and Lymphatic Vessel Zip codes
•
Tumor blood vessels differ from vessels in normal tissues
- angiogenesis-related
- tumor type-related
- tumor stage-related
•
Tumor lymphatics differ from lymphatics in normal tissues
- tumor type-related
- tumor stage-related
•
Vascular and tumor extracellular matrix differs from normal
ECM
- Clotted plasma proteins
- ECM proteins
•
The markers of tumor vessels enable new therapies
- guided drugs
- druggable targets
•
Vascular zip code molecules are useful as guidance elements
in nanoparticle targeting
CREKA-IO nanparticles may extravasate from some tumor vessels
Simberg et al.
Homing peptide for Extracellular Matrix in Tumor Vessels
and Tumor Stroma
Tumor/FITC-CREKA
Tumor /FITC-CSG
Green= FITC conjugates; red=blood vessels; blue=nuclei
M. Essler, M., H. Biliran et al.,
Multistage tumorigenesis in RIP-Tag
transgenic mice
Normal stage
(onc+)
Hyperplastic/
Dysplastic
stage
Angiogenic
stage
Tumor
stage
<5 wks
5-7 wks
7-12 wks
12-14 wks
100%
~50%
~10%
2-4%
Modular Approach to
Multifunctional
Nanoparticles
Physically target:
- vascular cells
- tumor cells
- extracellular matrix
Trigger/engineer:
- tissue penetration
- RES avoidance
- subcellular targeting
- diagnostic signal(s)
- release of therapeutic agent
- amplification of targeting
Ni-liposomes reduce RES uptake of CREKA-IO nanoparticles
D. Simberg, T. Duza et al.
Treatment of mice with decoy Ni-liposomes produces only
minimal background in control organs
D. Simberg, T. Duza et al.
Heparin pretreatment, but not platelet depletion, inhibits CREKA-IO-induced
clotting in tumor vessels
D. Simberg, et al.