Transcript Combination Nanopreparations And Intracellular Targeting For Mdr

COMBINATION NANOPREPARATIONS
AND INTRACELLULAR TARGETING FOR
MDR TUMORS
Vladimir Torchilin, Ph.D., D.Sc.
Center for Pharmaceutical Biotechnology and
Nanomedicine, Northeastern University, Boston, MA
02115, USA
Moscow, November 16-17, 2011
Why do we need a pharmaceutical carrier?
1. Protect a drug from the body
2. Protect the body from the drug
3. Adjust pharmacokinetics, i.e. distribution
and clearance
PHARMACEUTICAL NANOCARRIERS
soluble polymers
cyclodextrines
microcapsules
dendrimers
microparticles
carbon nanotubes
cells
fullerenes
cell ghosts
drug nanocrystals
liposomes
gold nanoparticles
micelles
Ca phosphate
niosomes
silica nanoparticles
solid lipid particles
lipoproteins
Pharmaceutical nanocarriers of choice
An ideal pharmaceutical nanocarriers for i.v adminstration
 Biocompatible & Biodegradable
 Small size, high loading capacity
Liposome
 Prolonged circulation
 Tumor accumulation
Polymeric Micelle
Hydrophil
ic
Drug
block
s
Hydropho
bic
block
Key issues in drug delivery to
tumors (MDR):
1. Get inside cells
2. Bypass/overcome resistance
TARGETING
Surface binding of mAb 2C5 to different human
tumor cell lines as shown by flow cytometry
Cells only,
Fluorescein-conjugated goat anti-mouse IgG
Isotype matched control antibody UPC10,
mAb 2C5
Modification of Liposomes with mAb 2C5
CH3(CH2)m
O
CH2
CH3(CH2)m
O
CH
CH2
O
O
P
O
O
O
CH2CH2NH
C
O
(C H2CH2O)n C
NO2
O
O-H
pNP -P E G -P E
+
NH 2
L ig and
aqueous buffer, pH 8-9.5
CH3(CH2)m
O
CH2
CH3(CH2)m
O
CH
CH2
O
P
O-H
O
O
O
O
CH2CH2NH
C
O
(C H2CH2O)n C
NH
L ig and
Cytotoxic effect of IgG-Doxil® and mAb 2C5Doxil® on 4T1 cells
A
100
B
100
60
Viable cells (%)
Cell viability (%)
80
2C5-Doxil
IgG-Doxil
Doxil
2C5-Liposomes
No treatment
40
20
P<0.001
50
0
0
0
50
100
150
200
Concentration (μg/ml)
Murine 4T1 cells. A – Cytotoxic effect of different concentrations of IgG-Doxil® (■)
and mAb 2C5-Doxil® (●) on 4T1 cells, and B – cytotoxicity of various preparations at
the fixed concentration of IgG-Doxil® and 2C5-Doxil® (as 100 µg/ml free
doxorubicin) and same concentration of 2C5-liposomes (as lipid)
In vivo tumor accumulation of various 111In-labeled liposomal formulations in murine
Lewis lung carcimona (LLC) model (A) (n = 5, results indicated± SD); and whole body
gamma-imaging of murine breast 4T1 tumor-bearing mice, 6 hr after the injection with
2C5-modified 111In-labeled liposomes. Circles indicate tumor locations (B)
Therapeutic activity, expressed as tumor volumes (A) and
tumor weight (B) of 2C5-modified Doxil® against control
preparations in CT26 implanted mice
A
B
6
PBS
3500
5
IgG-modified Doxil
2C5-modified Doxil
2500
2000
* P  0.05- 0.005
1500
1000
*
500
*
0
0
10
20
Time (days)
*
*
30
Tumor weight (gm)
Tumor volume (mm 3)
Doxil
3000
4
3
2 P 0.05
1
0
2C5-Doxil® IgG-Doxil® Doxil®
PBS
Arrows indicate treatment schedule, 2 mg/kg/q 5 days. (n= 8-10), * Two way
ANOVA with Tukey’s HSD Post-Hoc test (Results indicated ± SD)
In vivo therapeutic efficacy of Doxil-loaded
liposomes against intracranial U-87 MG
astrocytoma xenograft in nude mice
Treatmen
t
Median
survival
time (days)
p-value
Untreated
25.5
-
LS-Doxil
44.5
p < 0.005
vs. untreated
25
IgG-ILSDoxil
41
p > 0.05
vs. LS-Doxil
0
2C5-ILSDoxil
71.5
p < 0.05 vs.
LS-Doxil and
IgG-ILS-Doxil
Percent survival
100
75
50
0
10
20
30
40
50
Time (days)
60
70
80
Preparation of immunomicelles
O
Micelle
CH2CH2 O
C
O
NO2
+
NH2
Ligand
aqueous buffer, pH 8-9.5
O
Micelle
- PEG-PE
O
O2N
O
C
O
- pNP-PEG-PE
CH2CH2 O
C
NH
Ligand
Paclitaxel-loaded PEG-PE-based
mAb 2C5-immunomicelles in vivo
6
5
4
3
2
1
0
Inhibition of Lewis lung
carcinoma growth in mice by
various preparations of
paclitaxel
free paclitaxel
plain micelles
2C5 - immunomicelles
2C5 - immunomicelles, muscle
plain micelles, tumor
2C5 - immunomicelles, tumor
tumor
Paclitaxell ( ng/g of tumor)
% dose/g of tumor
7
Tumor accumulation of
free paclitaxel
1250
1000
750
500
250
0
free paclitaxel
plain micelles
2C5-immunomicelles
Inhibition of tumor grow (%)
Tumor accumulation of
paclitaxel-loaded mAb 2C5immunomicelles in Lewis lung
carcinoma model
100
80
60
40
20
0
SILENCING
New system for effective stabilization and delivery of siRNA:
Reversible siRNA-phospholipid conjugate in PEG-PE
polymeric mixed micelles
1 2 3 4 5 6 7 8
Native siRNA
native
1:750
PE
PEG
siRNA
phospholipd
ta il
Left panel: Schematic structure of siRNA-PE/PEG-PE mixed micelles.
Right panel: stability of siRNA against nucleolysis in 1:750 mixed micelles
compared to that of the free siRNA at different time-points till 24 h
Epifluorescence micrographs of MCF7 (Human breast adenocarcinoma)
cells stained with Hoechst33342 nuclear stain. A,B: Cells exposed to Cy3siRNA-Lip/PEG2-PE micelles for 2 hours. C,D: Untreated cells. A,C: UV
channel, B,D: Red channel
Gene silencing of different formulations
containing a 84 nM concentration of siRNA
40
Cell viability on GFP-C166 after
a 48 h incubation of different siRNA formulations
100
83.30
84.00
30
% of cell viability
% of gene silencing
35
25
20
15
10
79.45
75
50
18.33
25
5
0
0
naked siRNA
Formulations
1:750
1_200
1_500
1_750
siRNA/lipofectamine
Formulations
Left panel: % of gene silencing induced in GFP-C166 cells (comparison between
naked siRNA and siRNA-PE in 1:750 mixed micelles formulation).
Right panel: cell viability in the presence of various siRNA-PE-containing PEG-PEbased formulations in comparison with same amount of siRNA used as the
Lipofectamine formulation.
P-glycoprotein expression
% increase in Geometric mean
240
*
190
140
90
40
-10
SKOV-3
SKOV-3TR
P-gp expression in SKOV-3 and SKOV-3TR cells as shown by FACS analysis.
*P<0.005.
Doxorubicin cytotoxicity in (A) resistant and (B) sensitive MCF-7 cells after
treatment with siRNA nanopreparations. MCF-7 resistant and sensitive cells were
treated with formulations prepared with siRNA targeting MDR-1 (siMDR). Cells were
treated with doxorubicin (1µg/mL) for 24, 48, 72 and 96 h and cell viability was
measured. Data are expressed as the mean ± SD (n=3).
B
120
Doxorubicin alone
Free siMDR-1
siRNA-MDR-1
DOPE-PEI complexes
siRNA-MDR-1
nanopreparation
100
80
60
40
20
Cell Viability (%)
Cell Viability (%)
A
80
60
40
20
MCF-7 SENSITIVE
MCF-7 RESISTANT
0
24 h
48 h
Doxorubicin alone
Free siMDR-1
siRNA-MDR-1
siRNA-MDR-1
DOPE-PEI complexes
nanopreparations
72 h
96 h
Doxorubicin incubation time
0
24 h
48 h
72 h
96 h
Doxorubicin incubation time
IC50 for Paclitaxel at 48 hrs (nM)
Multidrug resistance reversal
10000
*
α
1000
*
100
SKOV-3
SKOV-3TR
10
1
PCL alone
PCL
liposomes
XRPCL
liposomes
IC50 for paclitaxel in SKOV-3 and SKOV-3TR cells. Cells were incubated in
96-well plate 24 hrs before the treatment at 3000 cells/well. Cells were than
treated with different formulations at various concentrations. Concentrations
represents tariquidar and paclitaxel concentration. Error bars indicate mean +
S.D. * p<0.05. α IC50 for paclitaxel-loaded liposomes in SKOV-3TR cells
was not achieved with dose as high as 1000 nM of paclitaxel.
DELIVERY INSIDE CELLS
IN ORDER TO ENHANCE INTRACELLULAR DELIVERY OF DRUG- OR
DNA-LOADED PHARMACEUTICAL NANOCARRIERS, THE CARRIERS
CAN BE ADDITIONALLY MODIFIED WITH SO-CALLED CELLPENETRATING PEPTIDES
11-mer TAT-peptide:
TyrGlyArgLysLysArgArgGlyArgArgArg
12 Hours
18 Hours
24 Hours
A1
B1
C1
A2
B2
C2
A3
B3
C3
Raman images from MCF-7 cells inoculated with d-DPPC
liposomes for 12, 18, and 24 hours. C-D stretching
intensities are shown in red.
3 Hours
6 Hours
9 Hours
A1
B1
C1
A2
B2
C2
A3
B3
C3
Raman images from MCF-7 cells inoculated with d-DPPC
TAT-peptide modified liposomes for 3, 6, and 9 hours. C-D
stretching intensities are shown in red.
IN VIVO TRANSFECTION OF TUMOR CELLS USING DNA-LOADED
LIPOSOMES ADDITIONALLY MODIFIED WITH CELLOPENETRATING PEPTIDE
Fluorescent and light microscopic images of Lewis lung carcinoma tumor
a
c
e
b
c
e
a,b; Untreated control tumor. c,d; Tumor injected with 100ul liposome/GFP pDNA complex (72 hrs
post injection).
e,f; Tumor injected with 100ul TAT-Liposome/GFP pDNA complex (72 hrs post injection).
Tumor sections at 50x objective magnification
In vitro cytotoxicity study
• Paclitaxel (PCT) loaded PEG750-PE micelles with 2.5
mol% TATp-PEG1000-PE
PCT
MCF-7
PCT micelles
TAT PCT micelles
120
*
100
*
80
60
40
20
Per cent cell viability
Per cent cell viability
MDA-MB-231
120
100
80
PCT
PCT micelles
TAT PCT micelles
*
*
60
40
20
0
0
50nM
PCT concentration
50nM
5nM
PCT concentration
5nM
*P < 0.05
PCT loaded micelles modified with TATp exhibited more cytotoxicity than
free drug and micelles without TATp
Schematic for the design of the multifunctional DDS used in this study that
includes pH-cleavable PEG-Hz-PE (a), temporarily “shielded” TATp (b),
and monoclonal antibody (c) attached to the surface of DDS via pH-noncleavable spacer
a
c
c
a a
a a
b
a a
a a
b
Incubation at lowered pH
b
b
Removal of PEG chains
Targeting by target specific antibody
and/or long circulation
De-shielding of the “hidden” function
Binding of rhodamine-labeled TATp-liposomes to
H9C2 cells
TATp-liposomes
TATp-liposomes were
incubated for 20 min at pH 5
before transfer to H9C2 cells
TATp-liposomes with
9% PEG-5000
TATp-liposomes with
18% PEG-5000
IN VIVO TRANSFECTION OF LLC TUMOR CELLS WITH
TAT-MODIFIED PEG-LIPOSOME pGFP COMPLEXES
UPON INTRATUMORAL ADMINISTRATION
Non-Cleavable
Cleavable
2G4/TATp-bearing Lipoplexes
for In Vivo Gene Delivery to Ischemic Heart
Experimental Myocardial Infarction (EMI)
by left coronary artery occlusion
Occlusion (30 minites)
Reperfusion
Formulation injection (i.v.)
Circulation (30 minutes)
Reocclusion
Blue Dye injection (i.v.)
Heart harvest
In vivo accumulation of
2G4-modified TATp-lipoplexes
with Rh-PE
upon i.v. injection in EMI rats.
In vivo transfection of ischemic myocardium
with 2G4-modified TATp-lipoplexes loaded
with pGFP
Bright field
Fluorescence
Tresholded
The bright field and fluorescence microscopy of frozen tissue sections from hearts in rats
with experimental myocardial ischemia were shown. The upper images represent a
montage of a single section. The bright fluorescence was found only in the ischemic
region of the left ventricle while low fluorescence was observed in other regions.
NEW LIGANDS FOR CELLSPECIFIC AND
INTRACELLULAR TARGETING
Lipid
Binding
peptide
+
Liposome
PVIII protein
Amphiphilic polymer
(PEG-PE conjugate)
Micelle
Drug-loaded liposome or micelles targeted by the pVIII protein.
The hydrophobic helix of pVIII spans the lipid layer and
binding peptide is displayed on the surface of the nanoparticle.
The drug molecules are shown as hexagons.
Binding selectivity to target cells by FACS analysis
on Coculture Models
Coculture Target MCF-7 with Nontarget/Non-cancer C166-GFP
Rhodamin-phage liposomes
Red fluorescence intensity (FL2-H)
Control Coculture Nontarget/Non-cancer NIH3T3 with C166-GFP
Untreated
Rhodamin-phage liposomes
Red fluorescence intensity (FL2-H)
% Cell binding
Untreated
Cytotoxity induced by Doxil and
phage-Doxil
A
B
140
140
120
120
100
% cell survival
% cell survival
C
80
60
40
100
80
60
40
20
20
0
0
0
5
10
15
20
25
30
conce ntration of dox(ug/ml)
2 4 h tre a tme n t b y p h a g e d o xil
2 4 h tre a tme n t b y Do xil
2 4 h tre a tme n t b p h a g e p la in llip o so me
35
4
8
12
16
20
24
28
32
36
40
44
time point (h)
pha ge Doxil tre a tme nt
Doxil tre a tme nt
48
Light
FITC
Fiter
Merg
e
Plainliposom
es
Phageliposome
s
Fig 4. Cytosolic delivery of HPTS encapsulated by phage-liposomes. while much less green
fluorescence is observed in the case of plain liposomes, phage-liposomes treatment shows strong
fluorescence emission at the 494nm excitation, indicating that HPTS is released into the neutral
cytosol.
GETTING TO INDIVIDUAL
ORGANELLES
Scheme of lysosomal targenting by RhB-liposomes loaded with
5-dodecanoylamino fluorescein di--D-galactopyranoside
(C12FDG), a lipophilic substrate for the lysosomal galactosidase
Liposomes
endosome
Cell
Octadecyl-Rhodamine B
C12FDG
lysosome
6
p = 0.004
p = 0.02
5
4
1
200 g/ml
2
200 g/ml
FL1
3
20 g/ml
Fluorescent Intensity of FITC (FL1)
7
P-Lip
RhBLip
P-Lip
0
Cntrl
RhBLip
C12FDG
Flow cytometry analysis of lysosomal targeting by RhB-Lip loaded with C12FDG.
HeLa cells were incubated with P-Lip or RhB-Lip (20 and 200 µg/ml) loaded with
C12FDG (1.5 % mol/mol). After 4 h incubation, the cells were washed and incubated
for 20 h with culture medium. Fluorescence intensity of FITC (channel FL1) was
determined in a FACS (FL1:FL2 compensation is 0.5%). A total of 10,000 events
were acquired for each sample. Each data set is the mean ± SD for 2 samples.
Increased apoptosis in tumors by transferrin-targeted
ceramide-loaded liposomes
p = 0.013
600
400
0.4
0.2
C
PBS
TL-Cer free
PL-C6Cer
ee
PL
-C
6C
er
TL
-C
6C
er
fr
er
TL
-C
Days post tumor inoculation
PB
S
0.0
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
DOPI
p = 0.028
0.6
200
0
TUNEL
Tumor weight, g
800
B 0.8
PBS
TL-Cer free
PL-C6Cer
TL-C6Cer
p < 0.02
% of tumor volume increase
A 1000
TL-C6Cer
Co-localization experiment using mitotracker green and
rhodamine-labeled TPP-L and PL. TPP-L and PL had 3
mole % TPP-PEG-PE and PEG-PE respectively.
Comparison of co-localization
Cell line used. HeLa, Incubation-time. 90 min, Dose. 500 nM.
Hoechst
FITC
Mitotracker
Deep red
Merged
D-Ac-TPP
D-Ac
Dendrimer
BF
There is a significant accumulation of D-Ac-TPP to
mitochondria compared to unmodified dendrimers.
4T1 tumor treatment in mice with different
preparations of paclitaxel
1200
Control
PL-PTX
TPP-L-PTX
800
***
400
0
3
5
7
9
11
13
15
17
19
21
23
24
26
Average Tumor Volume (mm3)
A
Time (Days) post-injection