1. Study
of
Cationic
Polylactides
as
a
Gene
Delivery
System
to
Combat
Against
Prostate
Cancer
CE
498
Undergraduate
Research
and
Creative
Activity
David
Huang
April
26th,
2013

2. Introduction
What
is
prostate
cancer?
Cancer
of
the
prostate
gland
Effects:
Impotence
and
infertility
Incontrollable
urine
flow
Weakening
of
bone
structure
Death
Angiogenesis
Cause
of
growth
of
aggressive
tumors
Provides
oxygen
and
nutrition
Stimulators
induce
angiogenesis
Interleukin-­‐8
(IL-­‐8)
Vascular
endothelial
growth
(VEGF)
Transforming
growth
factor
(TGB)-­‐β

3. Introduction
What
is
a
polylactide
(PLA)?
Polymer
of
lactic
acid
and
other
derivatives
Main
monomers:
lactic
acid
and
cyclic
di-­‐ester
(lactide)
Eco-­‐friendly
material
Biodegradable
Derived
from
renewable
resources
Form
typically
by
ring-­‐opening
polymerization
with
metal
catalysts
Wide
Range
of
Uses
Medical
supplies:
degradable
stitches,
screws,
pins,
rods
Compostable
packaging
Clothes

4. Gene
Therapy
Use
of
gene
therapy
to
combat
cancer
Recent
popularity
High
specificity
siRNA
used
to
silence
molecular
pathways
Cons
of
gene
therapy
Inadequate
cellular
absorption
Small
retention
time
in
body
Susceptible
to
breakdown
Needs
delivery
system

5. Use
of
Well-­‐Defined
Cationic
Polylactides
for
siRNA
Delivery
Protects
siRNA
from
biological
harm
Improve
cell
uptake
Positive
charges
of
cationic
polylactides
enhances
endocytosis
Adjustable
degradation
rate
Low
toxicity

6. Synthesis
of
Well-­‐Defined
Cationic
Polylactides
Achieved
using
organocatalyzed
ring
opening
polymerization
and
thiol-­‐ene
click
reaction
Step
1:
ROP
of
allyl-­‐functionalization
lactide
(LA)
with
L-­‐Lactic
Acid
Benzyl
alcohol
(BnOH):
initiator
4-­‐dimethylaminopyridine
(DMAP)
as
organocatalyst
Reaction
done
in
dichloromethane
(DCM)
Reaction
Conditions:
35
degrees
Celsius
Time:
One
week
90%
conversion

7. Synthesis
of
Well-­‐Defined
Cationic
Polylactides
Step
2:
UV-­‐induced
thiol-­‐ene
click
reactions
to
attach
tertiary
amine
group
Tertiary
amine
group:
2-­‐(diethylamino)ethanethiol
hydrochloride,
(DEAET)
Photo-­‐initiator:
2,2’-­‐dimethoxy-­‐2-­‐phenylacetophenone
(DMPA)
Reaction
Conditions
UV
irradiation
Room
temperature
Time:
30
minutes

8. Synthesis
of
Well-­‐Defined
Cationic
Polylactides
Adjust
[ene]0:[SH]0:[DMPA]0
ratio
to
produce
cationic
polylactides
with
different
amount
of
tertiary
amine
groups
Four
CPLAs
with
different
mole
%
of
amine-­‐polymer
backbone
units
Composition
determined
by
1H
NMR
CPLA-­‐9,
CPLA-­‐18,
CPLA-­‐30,
CPLA-­‐50
Suffix
number=mole
fraction

9. Confirmation
of
Well-­‐Defined
Cationic
Polylactides
Structure
1H
NMR
analysis
of
polylactide
made
in
first
step
1H
NMR
analysis
of
well-­‐defined
CPLA
made
in
2nd
step

10. Confirmation
of
Well-­‐Defined
Cationic
Polylactides
Structure
Gel
permeation
chromatography
Used
to
check
change
in
hydrodynamic
volume
between
PLA
and
CPLA
Results
of
GPC:
Showed
no
significant
change
in
volume
Concluded
no
crosslinking
Concluded
no
side
reactions

11. Study
of
CPLA’s
Degradation
Tests
done
at
samples
of
1.0
mg/ml
Tests
done
with
continual
GPC
analysis
Ran
at
two
temperatures
37
degrees
Celsius
25
degrees
Celsius
Data
collected
at
4
hour
intervals
from
1st
-­‐13th
hour
and
at
the
168th
hour

12. Study
of
CPLA’s
Degradation
Results
of
Tests
Faster
Degradation
Rate
at
37oC
Faster
Degradation
Rate
at
higher
amine
mole
%

13. Study
of
CPLA’s
Toxicity
Tests
conditions
PC3
cells
treated
with
all
4
different
CPLAs
Time
incubated:
48
hours
• Tests
Results
– Low
toxicity
– Most
toxic
was
CPLA-­‐50
at
highest
dosage

14. Use
of
CPLA
for
Gene
Delivery
Successful
Nanoplexes
Formed
Nanoplexes
formed
by
electrostatic
interaction
CPLA/siRNA
mass
ratio
20:1
Time:
30
minutes
TEM
image
of
CPLA-­‐50-­‐IL5
siRNA
Nanoplex

15. Conclusion
Synthesizing
CPLA
with
low
toxicity
is
possible
Degradation
rate
can
be
altered
to
control
gene
release
Use
of
Well-­‐Defined
CPLA
with
encapsulated
IL-­‐8-­‐
siRNA
can
provided
an
alternative
treatment
for
prostate
cancer