Formation of smart nanocapsules for defined
slow or sudden release
Anna Musyanovych and Katharina Landfester
Max Planck Institute for Polymer Research, Mainz, Germany
CO
O
COO
CO
O
CO
O
Functionalized nanoparticles
from degradable and nondegradable materials
PCR
Capsules for hydrophilic
compounds
Bio-reactions with a
single molecule inside a
droplet
Criteria for “perfect” nanocarrier
Inert polymer, e.g.
polystyrene
Hydrophilic
surface
Other receptors,
e.g. cell death
receptor
or
Contrast agent,
e.g. MRI
Biodegradable
polymer, e.g.
polylactide
“Stealthness”,
e.g. PEG-chains
Fluorescent
marker
Release receptor,
e.g. pH-, T-, UVsensitive
Drug
Cell receptor
Principle of the miniemulsion process
Formulation
Formulationof
ofsmall
smalland
andstable
stabledroplets
dropletsby
byusing
usinghigh
highshear
shear(e.g.
(e.g.ultrasound)
ultrasound)
Reactions in confined geometries
Phase I
high speed
stirring,
ultrasound
reaction
Phase II
Narrowly
Narrowlydistributed
distributednanodroplets
nanodroplets
Size
Sizerange:
range:50-500
50-500nm
nm
1:1
1:1copy
copy
(nanoreactor)
(nanoreactor)
Growth of droplets
Ostwald ripening:
+
Coalescence:
diffusion of oil
through the water
phase
Suppression of Ostwald ripening:
Addition of a co-stabilizer with low
solubility in a continuous phase
collision and fusion
of
oil droplets
Suppression of coalescence:
Effective surfactants
CH3 (CH2)11
SO4- Na+
Sodium dodecylsulfate (SDS)
C 16H35
OCH2CH2
OH
50
Lutensol AT50
Force:
Force:Same
Samechemical
chemicalpotential
potentialin
ineach
eachdroplet
droplet
K. Landfester, Macromol. Symp. 2000, 150, 171-178.
Direct and Inverse miniemulsions
non-polar phase
and hydrophobe
H2O
surfactant
e.g. sodium dodecylsulfate (SDS)
cetyltrimethylammonium chloride
(CTMA-Cl)
polar phase
and lipophobe (e.g. salt)
cyclohexane
surfactant
block copolymer
e.g. poly[(ethylene-co-butylene)-b-(ethylene oxide)]
Polystyrene functionalized nanoparticles
Oil phase
Aqueous phase
Styrene
Oil-soluble initiator
Oil-soluble fluorescent dye
Water
Surfactant
Water-soluble comonomer: e.g.
PEG-acrylate, vinyl phosphonic
acid, aminoethyl methacrylate, etc.
COO COO
COO
+
NH3
+
O
CO
OH
O
CO
O
2
PO 3
O
CO
PO
3
2
PO
O
2
3
PO3 2
Langmuir, 2007, 23(10), 5367-5376.
2
O
3
NH3
+
OH
H3N
OH
+
NH3
+
NH3
OH
OH
O
PO
NH3
+
PO3 2
O
CO
O
+
NH3
+
NH3
O
CO
O
Oil-soluble comonomer: e.g.
acrylic acid, glycidyl methacrylate, etc.
OH
OH
Colloid Polym. Sci., 2009, (in press).
Polystyrene functionalized nanoparticles
Oil phase
Aqueous phase
Styrene
Oil-soluble initiator
Oil-soluble fluorescent dye
Water
Surfactant
Dispersion of magnetite
Water-soluble comonomer: e.g.
PEG-acrylate, vinyl phosphonic
acid, aminoethyl methacrylate, etc.
COO COO
COO
+
NH3
+
O
CO
O
OH
PO
3
2
PO
2
3
O
PO3 2
3
2
O
PO
NH3
+
OH
H3N
OH
+
NH3
+
NH3
OH
OH
O
CO
O
2
PO 3
O
CO
NH3
+
PO3 2
O
CO
O
+
NH3
+
NH3
O
CO
O
Oil-soluble comonomer: e.g.
acrylic acid, glycidyl methacrylate, etc.
OH
OH
J. Phys.Condens. Mat. 2003, 15, S1345-1362.
Encapsulation of materials in nanoparticles
One colloid particle per polymer particle:
CaCO3 in PS
Many colloid particles per polymer particle:
Fe3O4 in PS
100nm
250 nm
Macromol. Symp. 2000, 151, 549.
Macromol. Chem. Phys. 2003, 204, 22.
One aggregate per polymer particle:
Carbon black in PS
50 nm
Macromol. Chem. Phys. 2001, 202, 51-60.
Characterization of functionalized nanoparticles
Particle
Particlesize
sizeand
andsurface
surfacegroups
groupsdensity
densitycan
canbe
beadjusted
adjustedby
byvarying
varyingthe
thetype
typeand
and
amount
amountof
ofsurfactant/functional
surfactant/functionalmonomer
monomer
Poly(styrene-co-acrylic acid)
0.5 wt%, Dz=220 nm, 200 mg Lutensol AT50
2 wt%, Dz=165 nm
1 µm
1 µm
1 µm
0.5 wt%, Dz=170 nm
400 mg Lutensol AT50
Langmuir, 2007, 23(10), 5367-5376.
Particle - Cell interaction
HeLa
HeLacells
cells
30
a
3 wt% NH3+
25
b
nFL1
20
20 μm
20 μm
c
d
-
COO
0 wt% NH3
+
COO
+
NH3
CO
O
Surface
Surfacefunctional
functionalgroups
groupsdensity
densityinfluence
influencethe
thecellular
cellularuptake
uptake
CO
O
+
COOCOO
CO
O
O
CO
O
CO
+
NH3
NH3
+
+
H3N
NH3
+
NH3
15
10
5
0
20 μm
50 μm
15 wt% NH3+
20 wt% NH3+
Increase
Increaseof
ofsurface
surfacefunctional
functionalgroups
groupsamount
amount
Musyanovych A., et al. In „Clinical Chemistry Research“, Mitchem, B. H. and
Sharnham, C. L. (ed.); Nova Science Publishers, Inc., 2009, Chapter VI.
Biomaterials, 2006, 27(14), 2820-2828.
Bioactive multifunctional composite particles
Induced
Induceddrug
drugrelease
releaseof
ofthe
thetumor
tumornecrosis
necrosisfactor
factorTNF
TNF
Functional building block 1
(TNF nanocyte)
Functional building block 2
(Lipid layer)
Fluorescent amine
functionalized
particle
Funktional building block 3
(PEG-scFv = Ligand)
Funktional building block 4
(cleavable PEG chain)
J. Control. Release 2009, 137, 69-77.
Bioactive multifunctional composite particles
FACS
+
NH3
+
NH3
+
NH3
+
H3 N
free lipids/liposomes
lipid-encapsulated particles
non-encapsulated particles
Cryo-TEM
- specific
100 nm
J. Control. Release 2009, 137, 69-77.
- non-specific
+
NH3
Bioactive multifunctional composite particles
cleavage in cell
by enzyme
tumor necrosis factor becomes active
Polymer precipitation within a nanodroplet
Poly(L-lactide)
Poly(ε-caprolactone)
Water
Solvent
Water
Solvent
evaporation
Poly(lactide-co-glycolide)
Polymer
Hydrophobic
compound, e.g.
marker, drug, etc.
Particle
Particlesize
sizeand
andsize
sizedistribution
distributionmainly
mainlydepend
depend
on
onthe
theamount
amountand
andtype
typeof
of polymer
polymerused
used
Macromol. Biosci., 2008, 23(10), 5367-5376.
Biodegradable magnetite particles
Water
Water
Solvent
evaporation
Solvent
Polymer
Magnetite
Effect
Effectof
ofmagnetite
magnetiteamount
amount
6.7 wt%
20 wt%
50 wt%
Macromol. Chem.
Phys. 2009, 210, 961.
Degradation of nanoparticles
Release
Releaseof
offluorescent
fluorescentdye
dye
from
frompoly(L-lactide)
poly(L-lactide)
particles
particles(HeLa
(HeLacells)
cells)
CLSM
Release
Releaseof
ofmagnetite
magnetite
from
frompoly(L-lactide)
poly(L-lactide)
particles
particles(MSC)
(MSC)
TEM
The
Therate
rateof
ofpolymer
polymerdegradation
degradationmainly
mainlydepends
dependson
onthe
thetype
typeof
ofsurfactant,
surfactant,
molecular
molecularweight
weightand
andTTggof
ofpolymer
polymer
Macromol. Biosci., 2008, 23(10), 5367-5376.
Crystallization in Gelatin Microgels
Transfer
to H2O
X-linking
Gelatine in water droplets
Particle size: 220 nm
Cross linking with glutaraldehyde
Loading with
CaCl2+
crystallization
by Na2HPO4
Apatite
Ca10(PO4)6(OH)2 in
gelatin microgels
Biomacromolecules, 2008, 9(9), 2383. Adv. Funct. Mater., 2008.
Encapsulation of liquids in miniemulsion
Capsules via phase separation
hydrophobic oil
polymer
Styrene/ Hexadecane PMMA/Parfume
Demixing
Polymerization
and phase separation
500 nm
Final morphology depends on:
• the interfacial tension between three
different phases (polymer, continuous
phase, dispersed nanodroplet)
• kinetics of the polymerization vs
phase separation
• miscibility of the phases
Macromol. Chem. Phys. 2009, 210.
Langmuir 2001, 17, 908-917.
Capsules via polymer nanoprecipitation
in inverse miniemulsion
Solvent/Non-solvent
Non-solvent
Solvent
evaporation
Water
Water
Polymer
poly(L-lactide)
poly(L-lactide)capsules
capsules
200 nm
poly(ε-caprolactone)
poly(ε-caprolactone)capsules
capsules
200 nm
Macromol. Biosci. 2006, 6(1), 33-40
Capsules via reaction at the interface
Oil
Addition of M2
Crosslinking
reaction at the
interface
M1
Water, hydrophilic
compounds, e.g. salt,
contrast agent, DNA, etc.
Hydrophilic
monomer
O=C=N
HO
OH
O
R
R'
N=C=O
Polymeric
shell
M1:
Crosslinking:
M2:
Redispersion in Water
aqueous phase
Oil
+
C
or
H2N
O
NH2
R
R
O
H
N
C
O
R'
H
N
H
N
or
C
O
Polyurethane
n
C
O
R
H
N
H
N
C
R'
O
Polyurea
H
N
C
O n
Capsules via reaction at interface
Polyurethane
Polyurea
Crosslinked starch
200 nm
M1:
Crosslinking:
M2: R'
O=C=N
N=C=O
HO
C
O
R'
+
N=C=O
n
O
R
Hydrolysis:
H
N
OH
C
or
H2N
R
O
O
C
O
NH2
R'
H
N
H
N
C
or
C
O
R
O
n
H
N
C
O
R'
H
N
C
R'
H
N
C
O n
Polyurea
H
N
NH-COOH
C
n
H
N
O
Polyurethane
R
+ H2O
H
N
O
R'
NH2
+ CO2
Macromolecules 2007, 40, 3122.
n
Langmuir, 2009, (in press).
Capsules via reaction at interface
Anionic
Anionicpolymerization
polymerizationof
ofn-butylcyanoacrylate
n-butylcyanoacrylate(BCA)
(BCA)
CN
n
CH2
C
OH
CN
-
HO
CH2
CN
C
CH2
C-
CN
H+
HO
CH2
C
CN
CH2
CH
O=C
O=C
O=C
O=C
O=C
O
O
O
O
O
C4H9
C4H9
C4H9
C4H9
n -1
DNA- Marker
Oil
PBCA shell
400
200
100
Progr. Colloid Polym. Sci., 2008, 134, 120.
C4H9
Amount of dsDNA
inside the droplets
1000
600
dsDNA
(790 bp)
n -1
Amount of dsDNA
inside the PBCA
capsules
About 15% of
encapsulated DNA is
in a form of free
chains
Capsules via reaction at interface
Increase
Increaseof
ofn-butylcyanoacrylate
n-butylcyanoacrylateconcentration
concentration
Progr. Colloid Polym. Sci., 2008, 134, 120.
Capsules as nanocontainers
Reaction in the nanocapsule,
e.g. reduction of Ag+ to Ag
Hydrophilic Gd
complexes for magnetic
resonance imaging (MRI)
Biomedical application
H
H
O
O
H
H
H
O
H
Gd3+
3+
Gd3+ Gd
H
O
H
H
O
H
Macromol. Chem. Phys. 2007, 208, 2229-2241.
Gd3+
O
H
O
H
H
O
200 nm
H
H
H
O
H
H
Multiplication of dsDNA
inside the drolpets
PCR
50 nm
Macromolecules 2007, 40, 3122-3135.
Biomacromolecules 2005, 6(4), 1824-1828
Droplets as “Bioreactors”
Polymerase
PolymeraseChain
ChainReaction
Reaction
DNA
DNAtemplate:
template:286
286bp
bp
PCR
PCR––product:
product:135
135bp
bp
DNA- Marker
dsDNA
Oil + Surfactant
Primers
dNTPs
Taq DNA Polymerase
Taq 10x Buffer
Mechanical
stirring
1500 bp
600 bp
400 bp
200 bp
PCR,
several cycles
Ultrasonication
100 bp
Biomacromolecules 2005, 6(4), 1824-1828
Cellular uptake
Polyurea
Polyureacapsules
capsuleswith
with
fluorescein
fluorescein
taken
takenup
upby
byHeLa
HeLacells
cells
Crosslinked
Crosslinkedstarch
starch
capsules
capsuleswith
withrhodamine
rhodamine
taken
takenup
upby
byHeLa
HeLacells
cells
CLSM
CLSM
Langmuir, 2009, (in press).
Release
Releaseof
ofmagnetite
magnetite
from
frompoly(L-lactide)
poly(L-lactide)
particles
particlesin
inMSC
MSC
TEM
Release mechanisms
1. Slow release
• Diffusion from the nanocapsules:
release depends on the shell
thickness and type of polymer
2. Fast release
•„switch“ e.g. by temperature, pH
change, enzyme…
• Degradation of the nanocapsules:
release depends on (bio)degradibility
without azo compound
Incorporation of blasting agent inside the capsule
with azo compound
Macromol. Mater. Eng. 2007, 292, 1237-1244.
Summary
uptake by cells
Inert polymer, e.g.
polystyrene
Hydrophilic
surface
specifity to certain
cells
Other receptors,
e.g. TNF
or
Contrast agent,
e.g. MRI
Biodegradable
polymer, e.g.
polylactide
D
marker included
“Stealthness”,
e.g. PEG-chains
Fluorescent
marker
defined release
Release receptor, e.g. pH, T,
UV-sensitive, enzyme…
stable in blood stream
Drug
Cell receptor
encapsulation of
drugs
Acknowledgments
Grit Baier
(Cross-linked starch and PBCA capsules,
PCR)
Eva Rosenbauer
(Polyurea capsules)
Dr. Umaporn Paiphansiri
(Polyurethane capsules)
Markus Urban
University of Ulm
Prof. Paul Walther
Dr. Oliver Zimmermann
Dr. Juliane Weich
Institute of Cell biology and
Immunology, University of Stuttgart
Prof. Roland Kontermann
Sylvia Messerschmidt
(Polylactide composite particles)
MPI for Polymer Research
Anika Hamberger
Dr. Anitha Ethirajan
Dr. Daniel Crespy
Dr. Ingo Lieberwirth
(PMMA capsules with blasting agents)
Financial support:
DFG (SPP1259, 1166, SFB); Max Planck Society; EU
VW-Stiftung; Fonds der Chemischen Industrie; Degussa/Evonik
Landesstiftung BW; BASF; Bayer Materials; Clariant