Transcript Name of presentation - Annual Unither Nanomedical

Biodegradable Nanoparticles for Cancer
Therapy
Jamboor K. Vishwanatha, Ph.D.
Dean and Professor
Graduate School of Biomedical Sciences
Poly D,L lactide-co-glycolide (PLGA)
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Extensively investigated polyester
Numerous assets
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Release profile can be controlled
Nanoparticle size can be controlled
Capable of the capture of any therapeutic agent

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Hydrophobic (ATRA, doxorubicin, 5 fluorouracil)
Hydrophilic (DNA, protein, small molecules)
Potential for development of targeted or
combinational therapies
Very low immunogenicity and cytotoxicity
High transfection potential
Properties of PLGA
Hydroxyl
terminus
Carboxyl
terminus
Lactide
Glycolide
PLGA undergoes acid catalyzed hydrolysis to release
cellular metabolites of lactic and glycolic acid
PLGA nanoparticle size can be controlled through
variations in nanoparticle formulation conditions
Variations in lactic acid to glycolic acid ratios effect
the degradation profile of the polymer (release rate)
Degradation rate is also affected through variations
in the intrinsic viscosity (i.v.) of the polymer
Nanotechnology Applications

Gene Delivery

Chemotherapeutic Delivery
Gene Delivery: Formulation of DNA
loaded nanoparticles
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Traditional formulation is accomplished through the use
a W/O/W double emulsion solvent evaporation technique
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Our formulation parameters include the use of a nonsolvent.
The addition of a non-solvent accomplishes several
goals
 Minimization of shear forces
 Decreases in particle size
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What do these particles look
like?
A
A
C
B
C
Pictures of PLGA nanoparticles following completion of fabrication (Panel A; Size bar ~100 nm).
Panel B is a TEM image PLGA nanoparticles (Size bar ~500nm). Panel C is a TEM image of
antibody targeted nanoparticles. Here the nanoparticles appear translucent with colloidal gold
labeled anti-mouse antibody as the dark specks (Size bar indicates 100nm).
Nanoparticles are stable at 4oC indefinitely and easily resuspend in
isotonic buffers or cell media.
PLGA nanoparticle size
A
B
C
Figure 2: Formulation parameters and their effect on size of plasmid DNA loaded
nanoparticles. Through the optimal choice of solvent/non-solvent systems we can control the size
of nanoparticles produced. Panel A: Solvent: Chloroform, Non-solvent: Water; size range 100>1000 nm. Panel B: Solvent: Chloroform, Non-solvent: Ethanol; size range 100-400 nm. Panel C:
Solvent: Chloroform, Non-solvent: Methanol; size range 51-138 nm.
It is important to be able to control the ultimate size of the particles in order to
achieve optimal transfection of cells and cross physiological barriers (i.e. blood
brain barrier and nuclear pore complexes).
Intracellular Uptake
Nanoparticles labeled with Nile Red appear red and can be seen
within the cells after 1 hour of incubation.
Percent viable cells
Nanoparticle efficiency: Uptake and
transfection
110
100
90
80
70
60
50
40
30
20
10
0
0
A
BB
250
500
750
1000
1250
Dose in micrograms
Transfection ability and cytotoxic effects of nanoparticles. PLGA nanoparticles were dual loaded with
sulforhodamine 101 (red) and GFP plasmid DNA (green) and exposed to DU-145 cells. Four days
post-transfection cells were visualized under laser confocal microscopy. Greater than 90% of the
cells display transcription of GFP encoding plasmid DNA and cellular uptake of the nanoparticles
(panel A). Unloaded nanoparticles were evaluated for cytotoxic effects upon cells (panel B). Greater
than 90 percent cell viability at the maximal dose of 1 mg/ml can be seen.
We have also observed no cytotoxic effects on cells at concentrations up to
3 mg/mL
pDrive-sh AnxA2 loaded nanoparticles
can serve to mediate prostate cancer
cellular migration
A
B
C
24 hours
D
E
F
48 hours
Migration of DU-145 cells upon administration of plasmid DNA loaded nanoparticles and blank
unloaded nanoparticles. Transfection of DU-145 cells was performed for 4 days and visualized 24 and
48 hours after plating of the migration assay. Control cells are seen in panel A and D respectively.
There is a tremendous reduction in cellular migration of DU-145 cells treated with plasmid DNA loaded
nanoparticles (panel B and E). There is no effect upon migration when treated with blank unloaded
nanoparticles (panel C and F). At 48 hours cells have been counter stained with crystal violet to
enhance visualization.
Number of Cells (X1000)
pDrive-sh AnxA2 nanoparticles also
effect prostate cancer cellular
proliferation
150
100
50
0
0
1
2
3
4 5 6
Time (days)
7
8
9
DU-145 cells were exposed to
nanoparticles over an 8 day time
course. Control cell growth is
indicated by the solid black line.
Unloaded blank nanoparticles
(dashed line) display no effect
upon cell growth or growth rate.
pDrive-sh AnxA2 loaded
nanoparticles (dotted line)
significantly diminish cellular
growth and rate of growth.
In vivo analysis of nanoparticle efficacy
A
A
B
C
Control HBSS
treated:
pDrive-sh AnxA2
nanoparticle treated:
18 days
27 days
Blank unloaded
nanoparticle
treated:
9 days
C
Control
pDrive-sh AnxA2
Blank
1000
750
500
250
0
0
10
20
30
Time (days)
40
50
Percent survival
Tumor volume (mm^3)
B1250
110
100
90
80
70
60
50
40
30
20
10
0
Control
pDrive-sh AnxA2
Blank
0
10
20
30
Time (days)
40
50
In vivo continued
Tumor progresison in male nude mice
1600
R2 = 0.9742
Tumor volume (mm^3)
1400
1200
sh
1000
con
R2 = 0.9574
blnk
800
Linear (blnk)
600
R2 = 0.9138
400
Linear (con)
Linear (sh)
200
0
0
5
10
15
20
25
Time (days)
Control 21 days
Sh treated 27 days
Blank 9 days
Chemotherapeutic Delivery:
‘Nanocurcumin’
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Polymeric nanoparticles encapsulating
Curcumin (anti-cancer drug)
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Curcumin:
Diferuloylmethane, a yellow polyphenol
extracted from Curcurma longa
 Therapeutic agent in traditional Indian
medicine
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Curcumin Vs Nanocurcumin
Free Curcumin
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Poorly dispersible
in water
Reduced
Bioavailability
Nanocurcumin
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Dispersible in water
Sustained drug release kinetics
Improved Bioavailability
Improved cellular uptake
Improved inhibition of
clonogenicity of cancer cell
lines
Characterization
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Percent Yield : 90-94
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Encapsulation Efficiency: > 95%
Particle Size Analysis:
Formulation Optimization
Batch
PVA
Conc.
Sonication
Time
Particle Size
range
Percent
Percent
Yield
Encapsulation
A
1.5%
1.0 min
150-250 nm
90.00
93.73
B
1.5%
2.0 min
100-200 nm
92.78
94.60
C
2.0%
2.0 min
20-100 nm
92.01
90.88
Surface Morphology:
500nm
Transmission Electron Microscopy
Confocal Microscopy:
Curcumin
PLGA Nanoparticles
Curcumin nanoparticles were observed under Confocal
Microscope (Carl Zeiss LSM 410). For curcumin: λex is 450nm
and λem is 488nm
In-vitro Release Kinetics
% Cuucumin release
75
60
45
30
15
0
0
50
100
150
200
250
Time (h)
Curcumin nanoparticles were incubated in PBS (pH 7.4) and at different
time points, the supernatant was analyzed at λ:450nm for cumulative
curcumin release
Cell Viability Assay
1.2
Cell viability (Rel)
Cell viability (Rel)
1.2
0.8
0.4
LNCaP
0.8
0.4
PC3
0
0
0
10
20
30
0
40
10
20
30
40
Curcumin (mM)
Curcumin (mM)
Control
Blank
Free curcumin
Nano curcumin
1.2
Cell viability (Rel)
Cell viability (Rel)
1.2
0.8
0.4
PWR1E
0.8
0.4
SKBr3
0
0
0
10
20
Curcumin (mM)
30
40
0
20
Curcumin (mM)
40
Second generation nanoparticles
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We are working on the development of
targeted nanotherapeutics
The goal of our work is to deliver locally
higher concentrations of drug to diseased
cells or tissues
These nanoparticles are capable of selective
attachment of nucleophilic substrates
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Antibodies
Proteins (Transferrin)
Peptides (NLS sequences)
Small molecules (N-acetyl cysteine)
Schematic diagram for the development of
targeted nanoparticles
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Using a platform technology we first generate an activated
nanoparticle
In a second reaction the targeting agent is conjugated to the
outer surface of the nanoparticle
Mode of action
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The targeted nanoparticle finds the specific cellular target
The nanoparticle binds to the surface of the cell
 If the target is internalized (i.e. folate receptors) the
nanoparticle is carried to the intracellular environment
 If the target is not internalized (i.e. annexin A2) the delivery
system has been engineered to release the nanoparticle at
the surface of the cell allowing for endocytosis to occur
PSMA targeting under co-culture
conditions
Activated nanoparticles loaded
with sulforhodamine 101 (red)
were quenched and exposed to
PSMA antibody. Following 1
hour, untargeted nanoparticles
were exposed to a co-culture of
PC-3 and LNCaP C4-2 cells
under dymanic motion
conditions for 30 minutes. No
preferential uptake of
nanoparticles is observed.
Targeted preferential uptake
PSMA targeted nanoparticles
were loaded with
sulforhodamine 101 (red) and
exposed to a co-culture of PC3 and LNCaP C4-2 cells for 30
minutes under dynamic motion.
Samples were fixed in
paraformaldehyde and
visualized through laser
confocal microscopy.
PC-3 cells are shown with yellow arrows, LNCaP C4-2 cells are shown
with green arrows. It is evident that there is a preferential uptake of
targeted nanoparticles to the LNCaP C4-2 cell line.
Anatomy of the eye
Intravitreal
injection
Potential convective current for vitreous
Nanoparticles are capable of reaching the retinal
cell layers
Pig retinal section 4 days post-intra vitreal
injection of nanoparticles. Nanoparticles
were loaded with sulforhodamine 101 (red)
and GFP plasmid DNA (green). The
concentration of nanoparticles was 1 mg/75
mL. Nuclear visualization was performed
using hematoxylin. The section shown is
located in the posterior portion of the retina.
Ganglion cell layer
Outer Nuclear Layer
Inner Nuclear Layer
Retina
Investigation of the ciliary body after intravitreal injection of nanoparticles
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Confocal image of the ciliary body from pig retinal
sections. It appears that a higher accumulation of
nanoparticles is occurring.
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The ciliary body is
located adjacent to the
lens in the anterior
portion of the eye
One of the functions is
the production of vitreal
fluid
It may be possible to use
accumulation in the ciliary
body as a drug reservoir
for sustained release
Issues of drug transport
to the retina still remain
Reduction of reactive oxygen species in
various disease states
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We are developing multi-phase nanoparticles for
protection against oxidative damage to cells.
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We expect these nanoparticles to provide an
immediate scavenging response to cellular oxidative
stressors (first phase).
In the second phase we are going to provide
sustained long-term protection against oxidative
damage.
We anticipate applications of these nanoparticles in
the areas of glaucoma, ischemic recovery (stroke
victims) and COPD.
Preliminary data suggest that we are able to reduce
the effective dose of a known protective agent by 25
fold.
Protection of retinal ganglion cells from
reactive oxygen
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Control
IAA
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N-acetyl cysteine
Nanoparticle
IAA is an chemical
inducer of reactive
oxygen. Treatment
was with 8 mM
N-acetyl cysteine was
administered at a
concentration of 5 mM
N-acetyl cysteine was
conjugated to the
surface of the
nanoparticle at a
concentration of 0.5
mM
Visualization was
performed 20 hours
after IAA induction
Acknowledgements:
 Dr. Arthur Braden
 Dr. Anindita Mukerjee
 Mallika Valapala
[email protected]