Nanotechnology
for TumorTargeted Drug
and Gene
Delivery
Mansoor M. Amiji, PhD
Department of Pharmaceutical Sciences
and the Nanomedicine Education and
Research Consortium (NERC)
Northeastern University
110 Mugar Life Sciences Building
Boston, MA 02115
Email: m.amiji@neu.edu
Early
Disease
Detection
Targeted
Therapy
Nanotechnology Platforms
Organic Nanostructures
qPolymeric nanoparticles
qLipid systems (liposomes, emulsions)
qSelf-assemblies (micelles, etc)
qDendrimers
qCarbon nanostructures (nanotubes,
fullerenes)
Inorganic Nanostructures
qMetal nanoparticles and nanoshells
qSilicon nanostructures
qNanocrystals (quantum dots)
Tumor Microenvironment
Tumor Mass
Ref: R.K. Jain. Scientific American, 271: 58-65 (1994).
Pore Size Cut-Off of Tumor Blood Vessels
Representative electron micrographs
of MCa IV tumor vessels grown in the
dorsal chamber
The vascular pore cutoff size for six different
types of tumors grown in the dorsal chamber
(A) and four tumors grown in the cranial
window (B) was evaluated
Ref. Hobbs, S.K. et al. Proc. Natl. Acad. Sci. USA 95, 4607-4612 (1998).
Passive Delivery Strategies
Physical approaches
qSize (less than 400 nm in diameter).
qSurface charge (positive particles have longer tumor
residence time).
Chemical approaches
qpH responsiveness.
qRedox.
qSurface modification with PEG/PEO.
Active Delivery Strategies
Surface Attachment of
Targeting Ligand
qLow molecular weight
ligands (folate,
thiamine, sugars)
qPeptides (RGD, LHRH)
qProteins (transferrin,
antibodies, lectins)
qPolysaccharides
(hyaluronic acid)
Polymeric Nanoparticles
q Polymers offer a versatile platform for designing
nanocarrier systems (hydrophilic, hydrophobic,
biodegradable, charged, etc).
q Fabrication methods are simple, reproducible, and
can be scaled-up according to cGMP (e.g., Abraxane®).
q Nanoparticles offer large area-to-volume ratio for
maximum payload capacity.
q Surfaces can be modified for passive or active
targeting.
q Stability of the payload is improved upon
encapsulation.
q Release profile of the drug can be controlled based
on polymer properties (sustained release, pH-sensitive
release, etc).
Polymeric Nanoparticles for TumorTargeted Delivery
Weight %
PEO-PCL
Coulter Analysis
Weight %
PEO-PBAE
Size (nm)
Size (nm)
SEM Image
Time-Dependent Uptake and Distribution of
Nanoparticles in Breast Cancer Cells
PEO-PCL
DIC
Fluorescence
30 minutes
10 minutes
Fluorescence
60 minutes
60 minutes
30 minutes
15 minutes
DIC
PEO-PBAE
PEO-PCL nanoparticles, encapsulated with fluorescent dye and PEO-PBAE nanoparticles
encapsulated with Oregon Green-paclitaxel were internalized by non-specific endocytosis,
followed by vesicular transport in the cytosol. After 1 hour, majority of the nanoparticles were
localized in the cytosol and in the vicinity of their target.
Intracellular Paclitaxel Concentrations upon
Administration in Nanoparticle Formulations
Cellular PTX Concentrations Upon Continuous Exposure
0.9
0.8
0.7
0.6
0.5
Paclitaxel in Aqueous Solution
0.4
Paclitaxel in PEO-PBAE Nanocarriers
0.3
Paclitaxel in PEO-PCL Nanocarriers
0.2
Dose: 1 µM
0.1
0
0
2
Time (h)
4
6
Concentration of Paclitaxel in Cells (µM)
Concentration of Paclitaxel in Cells (µM)
Cellular PTX Concentration After Acute Exposure
0.45
Paclitaxel in Aqueous Solution
0.40
Paclitaxel in PEO-PBAE Nanocarriers
Paclitaxel in PEO-PCL Nanocarriers
0.35
0.30
0.25
0.20
0.15
0.10
0.05
Dose: 1 µM
0.00
0
1
2
3
4
5
6
Time (h)
Tritiated [3H] paclitaxel was administered as control aqueous solution or in PEO-PCL and PEOPBAE nanoparticle formulations in MDA-MB-231 breast cancer cells. For acute exposure, the
formulation was washed away after 6 hour incubation and the drug concentrations in the cells
were measured over time. For chronic exposure, the formulation was left in the culture
environment and the cells were periodically analyzed for drug content.
Cytotoxicity of Paclitaxel upon Administration
in the Nanoparticle Formulations
Cell-Kill Efficiency of PTX After Short Term Exposure
Drug Exposure Time = 6 hours
Cell-Kill Efficiency of PTX After Long-Term Exposure
120
Drug Exposure Time = 6 days
120
100
Percentage Cell Viability
Percentage Cell Viability
110
100
90
80
Paclitaxel in Aqueous solution
70
Paclitaxel in PEO-PCL Nanocarriers
Paclitaxel in PEO-PBAE Nanocarriers
80
60
40
Paclitaxel in Solution
60
Paclitaxel in PEO-PCL Nanocarriers
Paclitaxel in PEO-PBAE Nanocarriers
50
0.001
0.01
0.1
Log [Paclitaxel Concentration (µM)]
20
1
10
0
0.001
0.01
0.1
1
10
Log [Paclitaxel Concentration (µM)]
Paclitaxel was administered as control aqueous solution or in PEO-PCL and PEO-PBAE nanoparticle
formulations in MDA-MB-231 breast cancer cells. For short-term exposure, the formulation was
washed away after 6 hour incubation and the cell viability was determined. For continuous exposure,
the formulation was left in the culture environment for up to 6 days and the cell viability was
measured using MTS assay.
Nanoparticle-Mediated In Vivo Drug Delivery in
Human Cancer Xenograft Models
Paclitaxel in SKOV-3 Xenograft Model
Tamoxifen in MDA -MB -231 Xenograft Model
SKOV-3
35
Paclitaxel Concentration in Tumor Mass
(µg/g of tissue)
40
Plain Injection
%Total activity recovered per whole tissue
NP with no stabilizer
30
NP with F-68
NP with F-108
25
20
15
10
5
0
1 hr post-injection
6 hrs post-injection
Paclitaxel in Solution
Paclitaxel in PEO-PBAE Nanocarriers
Paclitaxel in PEO-PCL Nanocarriers
5
4
3
2
1
0
1 hour
5 hours
Time
Tritiated [3H]-tamoxifen or pacliatxel was encapsulated in PEO-PCL and PEO-PBAE
nanoparticles and intravenously administered to female Nu/Nu (athymic) mice bearing MDAMB-231 or SKOV-3 human cancer xenografts.
Therapeutic Efficacy of Nanoparticle-Mediated
Delivery in Human Cancer Xenograft Models
Tumor Volumes
Tumor Weights
200
Control
PTX Solution
PCL-PTX NP's
PbAE-PTX NP's
0.8
0.7
Tumor Weight (g)
Tumor Volume (mm3)
250
150
100
0.6
0.5
0.4
0.3
0.2
0.1
50
0
Control
0
5
10
15
20
25
PTX Solution
PCL-PTX NP's
PbAE-PTX
NP's
30
Time after Tumor Inoculation (days)
Paclitaxel was encapsulated in PEO-PCL and PEO-PBAE nanoparticles and administered
intravenously to female Nu/Nu (athymic) mice bearing SKOV-3 human cancer xenografts.
Intracellular Ceramide Modulation: Strategy to
to Overcome Drug Resistance
Synthesis
Metabolism
Enhancement of
Intracellular
Ceramide
Production
Inhibition of
Intracellular
Ceramide
Metabolism
Exogenous Ceramide
Administration
Multifunctional Nanoparticle Systems –
“Pack Hunting Strategy”
From Jurassic Park III: The Lost World
Multifunctional Nanocarriers for Reversal of
Drug Resistance in Cancer
Paclitaxel and Tamoxifen
Paclitaxel and C6 Ceramide
[paclitaxel]
[paclitaxel]
10 nM
120
10 nM
1 µM
Percent Cell Viability
Percent Cell Viability
SKOV-3
p<0.001
120
100
80
60
40
20
100
80
60
40
20
0
0
control
C6-ceramide
10 µM
paclitaxel
paclitaxel + C6ceramide
control
paclitaxel
paclitaxel +
tamoxifen
140
1 µM
p<0.001
10 nM
10 nM
p<0.001
100
80
60
40
20
0
Percent Cell Viability
Percent Cell Viability
SKOV-3TR
p<0.001
120
tamoxifen
15 µM
[paclitaxel]
[paclitaxel]
140
p<0.001
1 µM
p<0.001
1 µM
120
100
80
60
40
20
0
control
C6-ceramide
10 µM
paclitaxel
paclitaxel + C6ceramide
control
tamoxifen
15 µM
paclitaxel
paclitaxel +
tamoxifen
Paclitaxel, C6 ceramide, and tamoxifen were encapsulated in PEO-PCL nanoparticles
and administered to sensitive and resistant SKOV-3 ovarian carcinoma cells.
National Cancer Institute Announces $35 Million in
Awards to 12 Cancer Nanotechnology Platform
Partnerships
FOR IMMEDIATE RELEASE
Monday, October 17, 2005
National Cancer Institute Announces $35 Million in Awards to 12
Cancer Nanotechnology Platform Partnerships
The National Cancer Institute (NCI), part of the National Institutes of Health
(NIH), today announced funding for a major component of its $144.3 million, fiveyear initiative for nanotechnology in cancer research. Awards totaling $35 million
over five years, with $7 million total in the first year, will establish 12 Cancer
Nanotechnology Platform Partnerships.
[ Click here to read the full press release ]
Cellular Uptake and Trafficking of DNA Delivered
in Non-Viral Vectors
(A) DNA Complex Formation, (B) Uptake, (C) Endocytosis (endosome), (D)
Escape from Endosome, (E) Degradation (endosome), (F) Intracellular
Release, (G) Degradation (cytosol), (H) Nuclear Targeting, (I) Nuclear Entry
and Expression.
Ref: D. Luo and W.M. Saltzman. Nature Biotech., 18(1):33-37 (2000).
Nanoparticles for Targeted Gene Delivery
SEM Images
Control Gelatin
Coulter Analysis
PEGylated Gelatin
1.0 µm
1.0 µm
In Vitro Transfection of GFP-Expressing Plasmid DNA
EGFP-N1 plasmid DNA was administered in control and PEG-modified gelatin nanoparticles to
NIH-3T3 murine fibroblast cells. Periodically, transgene expression was measured qualitatively
with confocal microscopy and quantitatively with fluorescence-activated cell sorter.
In Vivo Transfection in Solid Tumor Model
Tumor expression of beta-galactosidase after nanoparticle-mediated intravenous and intratumoral
administration of pCMV-beta in Lewis lung carcinoma-bearing female C57BL/6J mice. Quantitative
results were obtained by enzymatic conversion of o-nitrophenol (ONP) from o-nitrophenol
galactoside (ONPG) by measuring the absorbance at 420 nm.
In Vitro Transfection of sFlt-1 Plasmid DNA
in MDA-MB-435 Breast Cancer Cells
Qualitative Expression (Western Blot)
Quantitative Expression (ELISA)
1
700
2
3
4
5
6
7
8
Gel
sFLt-1 Concentration (pg/ml)
600
SHGel
PEG-Gel
PEG-SHGel
500
Lipo
Naked Plasmid
400
300
200
100
0
0
1
2
3
4
5
6
7
8
9
Human sFlt-1 expression in the supernatant of cultured MDAMB-435 cells following 48 hours post-transfection with plasmid
DNA.
Lane 1 : Gel Nps encapsulated with pCDNA3-sFlt-1
Lane 2 : SH-Gel Nps encapsulated with pCDNA3-sFlt-1
Lane 3 : PEG-Gel Nps encapsulated with pCDNA3-sFlt-1
Lane 4 : PEG-SHGel Nps encapsulated with pCDNA3-sFlt-1
Lane 5 : Lipofectin complexed with pCDNA3-sFlt-1
Lane 6: Naked pCDNA3-sFlt-1
Lane 7: Untreated Cells
Lane 8: Recombinant protein sFlt-1
Time (days)
Soluble Flt-1 (sFlt-1) encoding plasmid DNA in control and nanocarrier formulations was
administered to MDA-MB-435 breast adenocarcinoma cells. The expression of sFlt-1 was
measured in the culture media using an ELISA and Western blot assays.
In Vivo Transfection and Therapeutic Efficacy
of sFlt-1 Plasmid DNA in Breast Tumor Model
CD-31 Stained
Tumor Volume Changes
Untreated
PEG-Gel Nps
PEG-SHGel Nps
120
Naked Plasmid DNA
100
80
60
40
20
0
0
5
10
15
20
25
30
PEG-S H-Gel Np
Tumor Volume (mm3)
140
Untreated Control
160
Time (days)
In vivo transfection and efficacy of sFlt-1 following intravenous administration of the
control and PEG-modified gelatin-based nanocarriers in MDA-MB-435 human breast
cancer bearing mice. The tumor volume changes were measured daily post-administration
of 60 µg total plasmid DNA dose. The microvessel density in tumor cryosections was
evaluated (shown with arrows) by CD-31 antibody staining
Engineered Multifunctional Nanoemulsions
Flaxseed Oil NE
Nanoemulsion
Safflower Oil NE
Particle Size
(nm)
Zeta Potential (mV)
Standard
91 ± 17
- 15.7 ± 8.9
Stearylamine
99 ± 10
16.0 ± 4.4
Deoxycholic Acid
134 ± 10
- 11.1 ± 6.0
Oral Bioavailability Enhancement of Paclitaxel
in Nanoemulsion Formulations
Percent Activity/gram of Tissue
6
Control solution
NE-Stearylamine
NE-Deoxycholic acid
NE-Standard
4
AUC (%h/g)
Formulation
Control Solution
Standard NE
Stearylamine NE
Deoxycholic acid NE
6.8 ± 0.99
15.9 ± 3.14
20.6 ± 3.01
33.5 ± 5.9
2
0
0
10
20
30
40
50
60
Time (hour)
3H-paclitaxel
encapsulation in nanoemulsions was dosed orally to C57BL/6J mice.
Plasma and tissue concentrations were measured from radioactivity values.
Delivery of Paclitaxel Across the Blood-Brain
Barrier in Nanoemulsion Formulations
Paclitaxel Conc.( µ g/gram)
4.5
4.0
1 hr
3.5
6 hr
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control Solution
Tween 80-NE
SA-NE
Nanoemulsion Formulations
3H-Paclitaxel
encapsulation in nanoemulsions was dosed intravenously in Balb/c
mice. Plasma and brain concentrations were measured periodically.
Gd-Nanoemulsions for Image-Guided Cancer
Therapy
Mouse Brain MRI Scan
T1 Relaxation Time Measurements
120
R1 = 9.2 mM -1sec-1
1/T1 (1/sec)
100
80
60
40
20
0
0
2
4
6
8
10
12
14
Gd Concentration (mM)
Gd-stearylamine nanoemulsions were prepared by complexing Gd with DTPA-PE and
then incorporating in the nanoemulsion systems at different molar concentrations. In the
in vivo studies, the formulation was administered intravenously to naïve mice.
120
Aq.solution
Nanoemulsion
100
ViableCells(%)
PTX Alone
Rhodamine-PTX
Paclitaxel and Ceramide Combination in Brain
Tumor Therapy: In Vitro Studies
80
60
40
20
0
1 nM
10 nM
100 nM
120
Aq.solution
ViableCells(%)
CER Alone
NBD-C6 Ceramide
Concentration of PTX
100
Nanoemulsion
80
60
40
20
0
1 uM
10 uM
Concentration of CER
ViableCells(%)
PTX + CER
Merged
120
Aq.solution
Nanoemulsion
100
80
60
40
20
0
1 nM + 1 uM
10 nM + 10 uM
100 nM + 10
uM
Concentration of PTX + CER
Metal Nanoparticles
Gold-Silver Alloy NP (Mole% Ag)
0
10
25
50
100
Gold-Gadolinium Alloy NP (Mole% Gd)
0
10
25
50
75
Plasmon Band
at 525 nm
Near IR Peak
at 800 nm
50 nm
Gold Nanoparticles
Gold Nanorods
Functionalization of Gold Nanoparticle Surfaces Using
Hetero-bifunctional PEG
O
'desymmetrization'
Ag2O, KI
OH-PEG-OH
CH2Cl2, 0oC, 30 min
n = 1.5 K
HO-PEG-OTs
K+ - S
O
Me,
MeOH, reflux, 48 hr
HO-PEG-S
Me
(90%)
(95%)
Coumarin-isocyanate
(85%) toluene, reflux
Me
O
O
O
N
H
O-PEG- S
Au
Coumarin-Functionalized
Gold Nanoparticles
Me
Au
+
Gold
Nanoparticles
TEM
Me
NaOMe / MeOH
then, Dowex DR 2030
O
O
O
N
H
O-PEG-SH
Coumarin-PEG Thiol
Fluorescemce
Nucleus
(76%)
O
O
O
O
N
H
O-PEG-S
Me
Superparamagnetic Iron Oxide-Gold Core Shell Nanoparticles
50-80 nm
Targeting
Ligand
PEG Spacer
Iron
Oxide
Core
Gold
Shell
Nanoparticle-Tagged RBC
(before magnetic field)
Nanoparticle-Tagged RBC
(after magnetic field)
< Approx. 60 nm iron oxide/gold core-shell nanoparticles with
about 5 nm gold shell.
< Surface modification, using hetero-bifunctional PEG, for
attachment of bioactive (targeting) ligands.
< Cell separation using magnetic field.
< Magnetic nano-sensors and nano-harvestors.
< Targeted MRI contrast agent (T 2 = 30 msec).
< Magnetic hyperthermia.
“….AT 9 O'CLOCK on the night of my first round of
chemotherapy, exactly six hours after I left the oncologist's
office wondering what all the fuss was about, my stomach
tumbled into my knees, my knees refused to work
altogether, and I crumpled to the floor in a clammy,
shivering heap.
I lay there until dawn, at one point vomiting on myself, at
another crying that I'd rather die of cancer than undergo
chemo again. I was hot. I was cold. My shoulders wouldn't
stop shaking. My legs wouldn't move at all. Huge
hallucinations rolled over me….”
Lynda Gorov May 2, 2006
Acknowledgements
u Financial Support
- National Institutes of Health (RO1’s and SBIR)
- National Science Foundation (IGERT)
- Foundations ( PhRMA, AACP, etc)
- Private Industries (Boston Scientific, Novavax, etc)
- Intramural Support (RSDF and URF)
u Research Collaborators
- Robert Langer, Vladimir Torchilin, and Michael Seiden.
- Nanomedicine Education and Research Consortium (NERC)
at Northeastern University.
u Trainees
- Post-Doctoral Associates
- PhD and MS Students
- Pharmacy Honors Students