Won Jong Kim, Ph.D
Department of Chemistry
POSTECH
Smart nanoparticles for drug, gene and nitric oxide delivery
2 BioMedical Polymer Laboratory
Drug Delivery System
Nanoparticle-based Delivery System
Various Drug Delivery Systems
Unfavorable side effects - Nonspecific distribution - Hair loss and immunodeficiency
Low efficiency - Poor solubility and stability - Renal/hepatic clearance
Multidrug resistance
- Active efflux of drugs
Limitations of small molecular drugs
Ref: Nat. Biotech. 2015, 33, 941-951.
Doxorubicin (DOX) Paclitaxel (PTX)
Nanomedicine (Targeted, Programmed)
3 BioMedical Polymer Laboratory
Self-assembly is a simple and useful mechanism for preparing nanostructure
in a complex biological system.
Tumor
Leaky vessel wall
Chemotherapeutic drug delivery system to target site
Nanostructures
Polymeric Micelles
Nanoparticles
Nanoconstruct DNA Hybridization
Host-Guest binding
Hydrophobic interacion
Self-assembled Nanostructures
4 BioMedical Polymer Laboratory
Biological Barriers of Self-assembled Nanomedicine
Renal clearance by glomerular filtration
MPS or RES by immune cells
such as macrophage
Extracellular Barriers Intracellular Barriers
Cellular Internalization by endocytosis
Endosomal escape
Cargo release
Embolization of capillaries
To overcome various biological barriers, stimuli-regulated intelligent delivery system is required
5 BioMedical Polymer Laboratory
Intelligent Drug Carrier: Stimuli-responsive
Stimuli-responsive intelligent system
• High accumulation on target site
• Site-specific release for low side-effect and high efficacy
Endogenous specific stimuli
pH Redox potential
Small molecule
Exogenous stimuli
> Tm
+
Temperature
Light
Ultrasound HIFU
Target stimulus
Target stimulus
6 BioMedical Polymer Laboratory
1. Drug delivery by active and passive tumor targeting
EPR effect (Tumor) Enzyme-responsive drug release
7 BioMedical Polymer Laboratory
Tumor targeting by EPR effect
Tumor
Blood vessel
Tum
or
Norm
al Tis
sue
• Most solid tumors show a “leaky” vascular structure in blood vessels due to rapid angiogenesis
• Nanomedicines can be delivered to tumor site by
abnormal molecular and fluid transport dynamics.
Ref: Cancer Res. 1986, 46, 6387-6392. EPR : Enhanced Permeability and Retention
8 BioMedical Polymer Laboratory
Paclitaxel (PTX) (or Taxol)
Natural chemotherapeutic drug
– Isolated from Taxus brevifolia (pacific yew) in 1971
Mitotic inhibitor
– Act by interfering in the normal microtubule growth during cell division
– Arrest cell at the G2-M phase of cell cycle
– Induce apoptotic death
Use in treatment of breast, ovarian, lung, head, neck cancer
Limitation: Poor water solubility
Ref: J. Med. Chem. 2006, 49, 7253-7269.
Paclitaxel
9 BioMedical Polymer Laboratory
Cyclodextrin (CD)
Cyclic oligosaccharides
– α-D-1,4-glucopyranose linked
– Produced from starch by means of enzymatic conversion
β-Cyclodextrin
Cyclodextrin Number of
glucose ring Height (nm)
Diameter of cavity (nm)
Diameter of exterior (nm)
α-cyclodextrin (α-CD) 6 0.79 ± 0.01 0.47-0.53 1.46 ± 0.04
β-cyclodextrin (β-CD) 7 0.79 ± 0.01 0.60-0.65 1.54 ± 0.04
γ-cyclodextrin(γ-CD) 8 0.79 ± 0.01 0.75-0.83 1.75 ± 0.04
Hydrophobic cavity
Hydrophilic exterior
10 BioMedical Polymer Laboratory
CD-PTX inclusion complex
Driving force
: Hydrophobic interaction
between the aromatic ring of PTX and the cavity of CD
Enhance the water-solubility of PTX
However ,
there is need further advance on it for intelligent drug delivery.
β-CD CD::PTX Inclusion complex
PTX
R. Namgung et al., Nat. Commun. 2014, 5, 3702.
11 BioMedical Polymer Laboratory
Poly(Maleic Anhydride)
Copolymer of maleic anhydride with another monomer
Contain active succinic anhydride groups in each repeating units
Graft molecules having hydroxyl group on the polymer
→ form degradable ester bond
→ render carboxylic acid group on polymer backbone by ring-opening
CD-OH or
PTX-OH
Poly(Maleic Anhydride) Poly-CDa or Poly-PTXb
X= CD or PTX
Ester bond
Polymer for Conjugating CDs and PTXs
R. Namgung et al., Nat. Commun. 2014, 5, 3702.
12 BioMedical Polymer Laboratory
Overall scheme
‘Conjugation’ plus ‘Inclusion-Complexation’
pCD pPTX pCD::pPTX
Nanoassembly
Released PTX
Hydrolysis of ester bond
Dissociation of inclusion
In collaboration with Prof. A. Hoffman (U of Washington) R. Namgung et al., Nat. Commun. 2014, 5, 3702.
13 BioMedical Polymer Laboratory
AP-1-targeting ligand-modified carrier
AP-1/pCD::pPTX Saline polyPTX Taxol pCD::pPTX AP-1/polyPTX
P<0.001
pPTX micelle: fast drug release,
but short-term effect
AP-1-pCD::pPTX: higher
stabilitysustained drug
releaselong-term effect
R. Namgung et al., Nat. Commun. 2014, 5, 3702.
14
BioMedical Polymer Laboratory
Programmed nanoparticle loaded nanoparticle for
deep penetrating three-dimensional cancer therapy
Won Jong Kim, Ph. D.
POSTECH
15
Size Dependent Biological Barriers in Drug Delivery System
J. Kim et al. J. Control. Release 2016, in press.
Kidney
Liver & spleen
Tumor tissue-specific accumulation
Normal cell
Ref. Clin. Cancer Res. 2012, 18, 3229-3241.
Cancer cell
Appropriate Size and Site-Specific Release for Efficient Delivery of Drugs
Stimuli-responsive drug release
Size control of nanoparticles
Small size ( < 15 nm)
Appropriate size ( 50 - 200 nm)
Non-specific cellular uptake
& Fast clearance
16
Overcoming Low Penetration into Tumor Tissue
Endothelial cell
Blood vessel
Small-sized nanoparticle (<15 nm)
- Fast cellular uptake
- Non-specific cellular uptake
Appropriate sized nanoparticle (100-200 nm)
- Tumor-specific uptake
- Low penetration ability
Limitation of deep penetration
Deep penetration of small nanoparticles
Introducing the concept of “Tumor-vector (100-200 nm)”
to carry bundles of small nanoparticles (<15 nm) with drugs
1st Stimuli
2nd Stimuli
Cellular uptake & drug release
17
“DNA-Driven Drug Delivery Carrier”
Tumor-vector equipped with three-stage propulsion motioned by DNA and LPMSN
Deep Penetration Drug Release
Cellular uptake
DNA transition
EPR Effect
Tumor tissue
DNA transition
DNA-AuNPs pH-responsive DNAs DOX Large pore MSNs (LPMSNs)
MSN : mesoporous silica nanoparticle
18
Strategy
19
Characterization of Tumor-vector
TEM image
Tumor-vector (via DNA hybridization)
5 0 n m5 0 n m
Si map O map Au map Si + Au map BF
Successful formation of tumor-vector via DNA hybridization
J. Kim et al. Advanced Materials, in press.
SEM image
20
pH-Responsive AuNP and Drug Release
0 2 4 6 8 10 12
0
20
40
60
80
100
pH= 6.5
pH= 7.4
Re
lea
se
d A
u-D
NA
(%
)
Time (h)
Centrifugation at 4000 rpm, 5 min Absorbance of supernatant
Au release at tumor microenvironmental pH
pH=6.5
Tumor tissue pH (6.5)
Neutral pH (7.4)
0 10 20 30 40 50
0
20
40
60
80
100
pH= 5.0
pH= 6.5
pH= 7.4
Cu
mu
lative
re
lea
se
(%
)
Time (h)
Centrifugation at 13200 rpm, 20 min Fluorescence of supernatant (ex 495, em 555)
DOX release intracellular endosomal pH
pH=5.0
Neutral pH (7.4)
Tumor tissue pH (6.5)
Intracellular endosomal pH (5.0)
Approximately 49 AuNPs in 1 MSN (in TEM image) The amount of loaded AuNPs and DOX was calculated by back-titration of the supernatant
J. Kim et al. Advanced Materials, in press.
21
In Vivo Antitumor Effect and Deep Penetration MDA-MB-231 cell xenograft i.v. injection (5 mg/kg DOX) n =6
The highest anticancer effect (EPR, deep penetration, pH-sensitive drug release)
0 5 1 0 1 5 2 0 2 5 3 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
D a ys a fte r in je c tio n (d )
Tu
mo
r v
olu
me
(m
m3)
S aline
F ree D O X
T r i- i-D O X
c T ri- i-D O X
T r i-c i-D O X
T r i- i
c T r i- i
***
**
**
Tumor growth inhibition
Dual-pH responsive
Without DOX
Single pH-responsive Free DOX
** P<0.01, *** P<0.001 J. Kim et al. Advanced Materials, in press.
22 BioMedical Polymer Laboratory
“Nitric Oxide Delivery System”
Stimuli-responsive NO delivery system
NO-responsive transforming hydrogel for biomedical applications
23 BioMedical Polymer Laboratory
NO in vivo
NO
Ref. Clin. Science 2000, 98, 507-520.
J. Kim. et al., J. Mater. Chem. B 2014, 2, 341-356.
In vivo production of NO via L-Arginine
NOS
L-Arginine N-Hydroxy-L-Arginine L-Citrulline
Highly reactive free radical molecule (t1/2<6s) Produced by a NOS (Nitric Oxide Synthase) in vivo.
24 BioMedical Polymer Laboratory
Biological Function of Nitric Oxide (NO)
Antibacterial activity
O2-
ONOO-
(peroxidation)
vasodilation
Smooth muscle cell relation
Cell proliferation
Neuro-transmission
Presynaptic
terminal
Postsynaptic terminal
NMDAR
Ca2+
CaM
NOS
NO
sGC
PKG
NO cGMP
EGFR
Ras
MAPK
Ref. Cell 1994, 79, 915-918.
NO is pathophysiological modulator of vasodilation,
cell proliferation, antibacterial activity, neurotransmission
25 BioMedical Polymer Laboratory
Platforms for Local NO delivery
Substrates
Surface coating
Monomer in PBS
(pH 8.5)
Biomedical
application
Thin layer NO installation
NO 80 psi
(NaOME/MeOH)
NO release
aq. sol.
(pH 7.0, 37℃)
1. NO-releasing hydrogels
2. Localized NO delivery Easy coating
Kim, J. et al., Bioconjug.Chem. 2011, 22, 1031-1038.
Kim, J. et al., Angew Chem Int Ed. 2013, 52, 9187-9191.
26 BioMedical Polymer Laboratory
Light-triggered NO delivery system
H+
1. Light irradiation
4. NO release
3. Degradation of coating layers
2. H+ generation
o-NBA
Diazeniumdiolates
NO
Light
irradiation Acid
generation
Acid-
mediated
degradation
NO
release
H+ o-NBA NO-donor NO Coating layers
27 BioMedical Polymer Laboratory
Corneal wound healing effects
28 BioMedical Polymer Laboratory
Corneal wound healing effects
0h 12h 36h 48h0
20
40
60
80
100
120
Siz
e o
f ep
ith
elial d
efe
cts
(%
)
PBS
pH@MSN-CaP
pH@MSN-CaP-NO
48 h 36 h 12 h 0 h
Siz
e o
f ep
ith
elia
l d
efec
ts (
%)
0
20
40
60
80
100
120 PBS pH@MSN-CaP
pH@MSN-CaP-NO
*
0h 12h 36h 48h0
20
40
60
80
100
120
Siz
e o
f ep
ith
elial d
efe
cts
(%
)
PBS
pH@MSN-CaP
pH@MSN-CaP-NO
Accelerated wound healing effects on the cornea at day 2 compared to that of other control groups
Collaboration with Kim, J. H. group in SNU.
C57BL/6 mice
Wound model: created by scraping corneal epithelium with a surgical blade
5 µL of 1mg/mL, 10 min light exposure, 4 times a day
Detection: 0.5% fluorescein sodium solution
Kim, J. et all. ACS Nano, 2016
29 BioMedical Polymer Laboratory
Nitric Oxide in Rheumatoid Arthritis
Ref. Nat. Rev. Rheumatol. 2009, 5, 543-548.
• NO may affect the pathogenesis of rheumatoid arthritis
• NO-responsive materials can target cell and eliminate over-expressed NO
Rheumatoid Arthritis Normal bone homeostasis Bone destruction in RA
Bone resorption
Bone formation
Osteoblasts
Osteoclasts
Bone resorption
TNF
IL-1β
IL-6
NO
Inflammation
NO Inflammation
Cartilage
damage
Up-
regulation
Macrophage
↑
30 BioMedical Polymer Laboratory
NO-Responsive Hydrogel
NOCCL : NO-cleavable crosslinker
N,N’-(2-amino-1,4-phenylene)diacrylamide J. Park et al. Advanced Materials, in press.
31 BioMedical Polymer Laboratory
Synthesis of NOCCL
NOCCL was successfully synthesized and confirmed by 1H NMR
J. Park et al. Advanced Materials, in press.
32 BioMedical Polymer Laboratory
Swelling Test in NO Solution
40% Acrylamide 0.0625% NOCCL 40% Acrylamide 0.0625% BIS
After 24h
0 20 40
1
2
3
4
5
1570 M
157 M
15.7 M
1.57 M
0 MRela
tive s
well
ing
rati
o
Time (h)
0 20 40
1
2
3
4
5
Rela
tive s
well
ing
rati
o
Time (h)
1570 M
157 M
15.7 M
1.57 M
0 M
1 2 3 4 5 1 2 3 4 5
1 cm 1 cm
NO-responsive hydrogel swelled gradually by the cleavage of NOCCL
N,N’methylenebisacrylamide (BIS)
J. Park et al. Advanced Materials, in press.
33 BioMedical Polymer Laboratory
Hydrogel Deformation by NO Gas
NO-responsive hydrogel deformed by NO gas
CTL
gel
2 min 4 min 6 min
8 min 10 min 12 min
14 min 16 min 18 min Injection of
NO or Ar gas
control
gel
NO-r
gel
0 min
control
gel
NO-r
gel
J. Park et al. Advanced Materials, in press.
Commercial plastic bucket (10cm x 13cm x 5 cm)
0.4 cm holes that allowed gas to enter and exit
34 BioMedical Polymer Laboratory
Hydrogel Deformation by NO Gas
Commercial plastic bucket (10cm x 13cm x 5 cm)
0.4 cm holes that allowed gas to enter and exit
A movie demonstrating the deformation of NO-responsive gel
J. Park et al. Advanced Materials, in press.
Control gel NO-r gel
35 BioMedical Polymer Laboratory
Enzyme-responsive gel transformation
Enzyme-responsive
NO donor
Encapsulation
NO-responsive gel
Swelling
Enzyme
36 BioMedical Polymer Laboratory
Swelling Test using NO Donor
β-gal
β-gal-pyNO( ) β-galactose
+ 2NO NO release
β-gal
pyNO
β-gal-pyNO-
trapped hydrogel NO generation
and gel swelling
NO donor : β-gal-pyNO
0 3 6 90
50
100
-gal
Esterase
Cu
mu
lati
ve
re
lea
se
Time (h)
0 3 6 90
200
400
600
800
1000
-gal
EsteraseNO
(p
mo
lm
g-1
s-1
)
Time (h)
NO releasing profile
Total 2.71 umol in 1mg
β-gal-pyNO can supply a sufficient amount of NO for cleavage of crosslinker J. Park et al. Advanced Materials, in press.
37 BioMedical Polymer Laboratory
Swelling Test using NO Donor
Enzyme-responsive hydrogel was successfully
obtained by encapsulation of enzyme-triggered NO
donor
0 20 400
1
2
3
4
5
Re
lati
ve
sw
ell
ing
ra
tio
Time (h)
-gal-pyNO 0.00 mg
-gal-pyNO 0.13 mg
-gal-pyNO 0.25 mg
-gal-pyNO 0.50 mg
-gal-pyNO 1.00 mg
NO-res. gel, β-gal+
0 20 40
1
2
3
4
5
6
Re
lati
ve
sw
ell
ing
ra
tio
Time (h)
gal-pyNO 0.00 mg
gal-pyNO 0.13 mg
gal-pyNO 0.25 mg
gal-pyNO 0.50 mg
gal-pyNO 1.00 mg
NO-res. gel, Esterase+
0 20 40
1
2
3
4
5
6 gal-pyNO 0.00 mg
gal-pyNO 0.13 mg
gal-pyNO 0.25 mg
gal-pyNO 0.50 mg
gal-pyNO 1.00 mg
Re
lati
ve
sw
ell
ing
ra
tio
Time (h)
control gel, β-gal+
NO-responsive
gel
control
gel
β-gal
Esterase
β-gal-pyNO
- + + - + + - - - + - - - + - + - +
J. Park et al. Advanced Materials, in press.
38 BioMedical Polymer Laboratory
Swelling Test in Macrophage Cells
-LPS +LPS -LPS +LPS
0
5
10
15
20
NO-responsive gel control gel
R
ela
tive
sw
ellin
g r
ati
o (
%)
RAW264.7
NIH/3T3
a
c
RAW 264.7 cells NO production
LPS
hydrogel
Gel swelling
NO NO
b
d NO-responsive
gel
control
gel
Cells
LPS - + + - + +
- - + - - +
NIH/3T3
RAW 264.7
**
NIH/3T3 RAW 264.7
0
20
40
NO
pro
du
cti
on
(M
)
LPS-
LPS+
**
400,000 cells
6 well plate
J. Park et al. Advanced Materials, in press.
39 BioMedical Polymer Laboratory
Swelling Test in Macrophage Cells
RAW 264.7 cells NO production
LPS
hydrogel
Gel swelling
NO NO
NO-responsive
gel
control
gel
Cells
LPS - + + - + +
- - + - - +
NIH/3T3
RAW 264.7
J. Park et al. Advanced Materials, 2017, 29, 1702859.
40 BioMedical Polymer Laboratory
Acknowledgements
Dr. Yeong Mi Lee
Dr. Gurusamy
Saravanakumar
Dr. Swapan Pramanick
Prof. Won Jong Kim Prof. S. K. Yoon (Catholic Unv.)
Prof. S. W. Kim (University of Utah)
Prof. J. H. Kim (SNU Medical School)
Prof. S. B. Jo (The Univ. of Mississippi)
Prof. I-K. Park (Chonnam Nat. Univ.)
Prof. C. Y. Heo (SNU Medical School)
Prof. H. Lee (KAIST)
Prof. B. H. Lee (KNU)
Prof. I. K. Kwon (Kyunghee University)
Dr. Jinhwan Kim
Junghong Park
Youngnam Kang
Dongsik Park
Donghyun Jang
Hyeongmok Park
Sooseok Im
Seonil Kim
Seokhee Jo
Sungjin Jung
Jaeryong Jo
Lab members
Co-workers
Jiwon Yeo
Kunho Kim
Jaehyun Park
Hyori Lee
Tae Jeong Kim
BioMedical Polymer Laboratory