Targeted Bioresponsive
Nanoparticles
Francis C. Szoka, Ph.D.
University of California
2006 Particles Meeting
Orlando, Fl
May 14, 2006
Happy Mothers’s Day
Is there’s plenty of room at the bottom?
10µm
Red blood cells
1µm
Dendrimers
Liposomes
Magnetic
nanoparticles
100nm
10nm
1nm
Aspirin
Quantum dots
Nature Biotechnology 21, 1161 - 65 (2003); Science 294, 1901 - 3 (2001); Ian Wark Research Institute
Summary
• Liposomes & Doxil™
• Non-biodegradable dendrimers
• Biodegradable dendrimer
Lipid Structure
Hydrophilic
Interface
Hydrophobic
Time Line for Liposome Drug
Carriers
•
•
•
•
•
•
•
Biomembrane 1965
Enzyme encapsulation 1971
Pharmacokinetics mid 70’s
Therapeutics late 70’s
Clinical trials 80’s
Initial approval early 90’s
Doxil™ approval 96
Drug Concentrations at a Tumor Site
Following I.V. Administration
Free drug
[C]
Carrierassociated drug
Free drug released from
carrier accumulated at
tumor site
Time
Structure of Doxil
Doxorubicin
85-100 nm
Lipid Membrane
(Phospholipid +
Cholesterol)
Polyethylene
Glycol
F.Martin
Doxil Morphology
100 nm
F.Martin
Plasma concentration (ng/mL)
DOXIL Pharmacokinetics
105
T½ = 2.5 d
4
10
103
102
T½ = 3.8 d
101
100
Metabolite appears in
few days
10-1
0
F.Martin
5
10
15
20
Time After Infusion (Days)
25
30
Serial Gamma Scintigrams of KS Patient after
Pegylated Liposomes Containing 111In-DTPA
4 hrs.
F.Martin
24 hrs.
48 hrs.
96 hrs.
What Might Further Improve
Liposome Drug/Gene Delivery?
• Cell targeting
– CD44 ligands
• Improved cytoplasmic delivery
– pH sensitive endosomal escape
• Control of surface properties
– Redox surface
Drug Carriers
• Limit drug entry into healthy tissue and cells
• Increase drug solubility, modify immunogenicity and enzymatic
degradation
• Passively target drug to tumors via “leaky”
vasculature (EPR effect)
- Tumor accumulation enhanced as size increases to 500 nm
Blood
capillary
small
diffusible
molecules
macromolecules
Normal
cells
Lymphatic
capillary
“Leaky”
capillary
No lymphatic
capillaries
Cancer cells
“Leaky”
capillary
Maeda, H., Seymour, L. W., and Miyamoto, Y. Bioconjugate Chem., 1992, 3, 351-362.
Dendritic Molecules for Drug
Delivery
Highly branched
• Multiple end-groups for drug attachment
• “Surface” functionality can be tailored
• Tunable solubility
• Decreased flexibility
Solubilizing group
Drug
Branching point
Targeting moiety
Well-defined, monodisperse < 10 nm
• Reproducible synthesis
• Reproducible pharmacokinetics
Lee et al., Designing Dendrimers for Biological Applications,
Nature Biotech. 23,1517, 2005.
POLYAMINES USED TO FORM
DNA POLYPLEXES
H2N
NH
NH2
NH
2
NH2
NH2
NH2
O
N
H O
N
H O
NH2
H O
N
N
H O
H O
N
NH2
N
H O
NH
2
H O
N
H2N
O NH
H
N
O
N
N
H
O
NH
2
POL
N
N
H
N
O
O
N
H
N
HN O
PAM
O
N
O
NH
H N
Non-buffering
Poor gene transfer
O
N
HN O
H O
N
HN
NH2
O
NH
NH2
HN
NH2
Strongly buffering
Good gene transfer
NH
‘PROTON SPONGE’
HYPOTHESIS H+
H+
B- B
BB-
pH
Cl-
Low buffering
DNA polyplex
pH
BB-
BB-
B-
Cl
High buffering
DNA polyplex
STRATEGY TO MEASURE
ORGANELLAR CHLORIDE
targeting
ligand
reference
Cl- sensor
Requirements of Cl- sensor:
bright, long-wavelength, ratioable
Cl- sensitive (0-100 mM), pH insensitive
stable, non-toxic... Sonewame, Szoka & Verkman, 2003
CALIBRATION OF A DEXTRAN
CHLORIDE SENSOR
BAC-dextran-TMR
BAC
10
solution
8
Fred
Fgreen
6
cells
4
TMR
2
0
20
40
60
[Cl-] (mM)
80
100
Cl
FLUORESCENT
SENSING
DNA-POLYAMINE POLYPLEX
DNA
BAC
BAC
BAC
dextran
BAC
BAC
dextran-BAC
H2N
S S
H2N
TMR
polyamine
NH2
TMR
polyamine-TMR
ENDOCYTOSIS OF DNA POLYPLEX
CHLORIDE SENSOR
0-1 min
15 min
30 min
60 min
75 min
BAC-dextran-TMR-polyamidoamine / DNA
ACIDIFICATION & Cl- ACCUMULATION
DEPENDS ON POLYAMINE BUFFERING
7.5 pH
120
[Cl-]
(mM) PAM
7.0
PAM
6.5
90
6.0
PEI
5.5
PEI
POL
60
POL
5.0
30
0
15
30
45
60
75
0
15
30
45
60
75
time (min)
time (min)
TMR-polyamine-CF-DNA
BAC-dextranTMR-polyamine-DNA
LYSIS/PRE-LYSIS IN PEI BUT
NOT POL ENDOSOMES
PEI
7.5
POL
disappeared
7.0
pH
6.5
6.0
5.5
5.0
0
15
30
45
60
75 0
15
time (min)
30
45
60
75
ENDOSOME SWELLING DEPENDS
ON POLYAMINE BUFFERING
endosome volume (μm3)
3.5
PAM
POL / chloroquine
2.5
1.5
POL
0.5
15
30
45
60
time (min)
75
‘PROTON SPONGE’
MECHANISM H+
H+
B- B
BB-
pH
Cl-
Low buffering
DNA polyplex
pH
BB-
BB-
B-
Cl
High buffering
DNA polyplex
A Spectrum of Dendritic
Architectures
dendrimer-star polymer hybrid
“bow-tie” polymer
dendronized hyperbranched
polymer
dendronized linear polymer
Polyester Dendrimers:
(
)n
(
)n
(
i
)n
O
O
Ph
O O
(
)n
i
ii
OH
(
ii
i
Ph
O
O O O O
O
O
O
OH OH
O
O
O O O O
O
Ph
O
O
Ph
i
ii
)n
OH
O
O
OH
O O O O
HO
O
O O
O O O
O O O
O O
O O
O O
Ph
Ph
OH
O O
O O
O O O
O O
OH
O
O
O
O O O O
Ph
ii
HO
HO
O O
O O O
Reagents:
i)
O DMAP
O
O
Ph
O
O
O O
(
HO
)n
O O
O O
OH OH
OH OH
OH OH
OH OH
O
Ph
ii) H2, Pd/C
Divergent synthesis with facile purification, high yields
Æ Resulting dendrons are degradable, non-toxic
Dendritic Molecules for Drug Delivery
(
(
(
(
(
(
•32 hydroxyls
•3790 Da
•Non-toxic
•t1/2 < 10 min
• 21,000 Da
• PDI = 1.02
• Non-toxic
• t1/2 = 72 min
• Liver accumulation
• Poor solubility of
drug conjugate at
high loadings
Padilla de Jesús and coworkers. Bioconjugate Chemistry 2002, 13, 453-461.
Dendritic “Bow-Tie” Polymers
Poly(ethylene oxide) (PEO)
• varying molecular weights
• sterically protects payload
Dendritic Scaffold
• biodegradable polyester
• dendron generation varies degree
of branching, drug loading potential
Gillies and Fréchet J. Am. Chem. Soc. 2002, 124, 14137.
Blood Circulation of “Bow-Tie” Polymers
Blood Pharmacokinetics in CD-1 Mice (~40 mg/kg)
(h)
[G-1]–10K (22 kDa)
8±1
33 (24 h)
[G-1]–20K (44 kDa)
1.4 ± 0.4
20 (24 h)
[G-2]–5K (23 kDa)
11 ± 3
22 (24 h)
[G-2]–10K (43 kDa)
26 ± 6
18
[G-2]–20K (87 kDa)
25 ± 8
10 (24 h)
[G-3]–5K (45 kDa)
31 ± 2
2
[G-3]–10K (85 kDa)
40 ± 4
2
[G-3]–20K (160 kDa)
50 ± 10
4
[G-1] - 10 K
60
[G-1] - 20 K
[G-2] - 5 K
50
[G-2] - 10 K
[G-2] - 20 K
% Dose / g blood
t1/2β
% Dose in
Urine (48 h)
Polymer & MW
40
[G-3] - 5 K
[G-3] - 10 K
30
[G-3] - 20 K
20
10
0
0
10
20
30
40
50
Time (h)
Summary • [G-3] polymers circulate longer, excreted slower than [G-2] polymers
• [G-1] polymers are rapidly cleared with most of the material in the urine
and feces after 48 hours.
Gilles et al. Mol. Pharm.2005
Similar Mass, Different Elimination
rates
[G-3]–5K (45 kDa)
31 ± 2
[G-1]–20K (44 kDa)
1.4 ± 0.4
Pores Restrict Reptation of Star
Polymers
Brochard-Wyart & de Gennes, 1996
Doxorubicin (Dox)
O
• Anthracycline antibiotic
OH
COCH2OH
OH
CH3O
O
OH
H3C
O
NH2
OH
O
• Dox has a wellestablished position in
the treatment of a variety
of human malignancies.
• Dox cardiotoxicity is a
major clinical handicap
limiting its cumulative
dosage.
Drug Concentrations at a Tumor
Site Following I.V. Administration
Covalent Attachment of
Doxorubicin
Synthesis Dendrimer Doxorubicin
Tumor Accumulation of “Bow Tie”
Polymers
Biodistribution of G3-5K at 24 and 48 h
30
24 Hours
48 Hours
20
15
10
5
ss
s
ce
ar
ca
C
Fe
rin
e
U
ea
rt
Lu
ng
s
Li
ve
St
r
om
ac
h
Sp
In leen
te
st
in
es
K
id
ne
ys
M
us
cl
e
Tu
m
or
H
d
0
lo
o
% Dose/g Tissue
25
B
• Biodistribution of
doxorubicin conjugate of
G3-5K (40 kDa)
performed in Balb/c mice
with subcutaneous C-26
tumors
• Tumor/muscle ratio of 16
at 48 h demonstrates
enhanced permeability of
tumor vasculature
Therapeutic efficacy of various
control formulations in C-26 colon
carcinoma in Balb/c mice.
Treatment Number Number Mean
P
animals survivors survival value
day
__
PBS
7
0
28
Acetone linked
Hydrazone Bowtie
238 mg/kg polymer
8
0
34
0.051
DOX carbamate
linked Bowtie
20 mg/kg DOX
10
0
30
0.019
C26 Tumor Progression
•
•
•
•
•
•
PBS
Doxorubicin 6 mg/kg
Bow-tie 1 mg/kg
Bow-tie 3 mg/kg
Bow-tie 6 mg/kg
Bow-tie 20 mg/kg
Effect of Bow-tie Dox or Doxil™ on Survival
Survival
10
9
8
7
6
5
4
3
2
1
0
Bow-Tie
20
mg/kg
6 mg/kg
3 mg/kg
1 mg/kg
0
10
20
30
40
50
60
Days
Survival
# of Mice
Doxil™
Doxil 20
mg/kg
10
9
8
7
6
5
4
3
2
1
0
Doxil 6
mg/kg
0
10
20
30
Days
40
50
60
Poly-Old
Linkage 20
mg/kg
Poly-Old
Linkage 6
mg/kg
Free Dox
6mg/kg
Current Status
• Liposomes-mature technology
– Excellent payload
– Biocompatible
– Large diameter
– Not suitable for all drugs
• Dendritric polymers-developing
technology
– Small diameter
– Biocompatible
– Suitable for most drugs
– Modest payload through chemical linkage
Si il
l
t f
i
d
ith
Nanoparticle delivery to the brain
Nanoparticles feature:
• Targeting
• Shielding
• Sensing
• Reporting
Pump
Lipid or
polymer
Convection Enhanced
Delivery
.
Red Blood Cell:
7 μm
• Convective flow increases
distribution to large volumes
• Direct tissue targeting
Nanoparticle:
50-100 nm
G. Huynh – Szoka
group
q
y
Surface Charge Modification
by Disulfide Exchange and
Ligand Insertion
HS-R’
DNA
Diacyl lipids
Chol-SSR (CTL)
PEG-lipid
Ligand
DNA
DNA
Chol-SSR’
TAT-PEGlipid
R, positive; R’, negative or neutral
TATpeptide-targeted NLP for
Brain Delivery
DNA
PEG-lipid
Chol-SSR (CTL)
TAT-PEG-lipid
DOPE
TAT-NLP Delivery to U87-MG Brain Tumor
No TAT ligand
NLP
GFP
Merge
High
intensity
Low
intensity
0.5 cm
With TAT ligand
1mm
(arrows: tumor border)
Intracellular drug transport
+
+
mi
+
c ro
tub
ule
+
+
+
+
+
nucleus
+
+
+
Biomolecular Adaptors
for Retrograde Transport
(BART)
+
+
cell
+
+
+
+
+
nucleus
membrane
Endogenous
dynein
Novel
Cargo
BART
peptide
+
ule
b
u
t
o
micr
Rich Cohen- Szoka group
Summary
• Self-assembled systems make good drug
carriers
• Improvements needed in targeting,
cytoplasmic delivery and surface
properties
• Sequential assembly, coupled with local
delivery, can improve gene transfer to
defined tissues
Acknowledgements
UCSF
UCB
• Professor Jean
• F. Szoka
Frechet,Ph.D.
• J. MacKay, Ph.D.
• Omayra Padilla, Ph.D.
• E. Dy
Funded by NIH
EB3008, CA107268
• Beth Gillies, Ph.D.
• Cameron Lee
Acknowledgements
•
•
•
•
•
•
•
•
•
Szoka group
J. MacKay
X. Guo
W. Li
Z. Huang
R. Eliaz
L. Gagne
C. Redemann
E. Dy
Many past
members
• D. Deen, Ph.D.
• Funded by NIH
EB3008,
CA107268
The Sequus
employees