Formation of smart nanocapsules for defined slow or sudden release

Anna Musyanovych and Katharina LandfesterMax Planck Institute for Polymer Research, Mainz, Germany

Bio-reactions with a single molecule inside a droplet

Functionalized nanoparticles from degradable and non-

degradable materials

Capsules for hydrophilic compounds

PCR

COO

COO

COO

COO

Hydrophilic surface

“Stealthness”, e.g. PEG-chains

Criteria for “perfect” nanocarrier

Inert polymer, e.g. polystyrene

Fluorescent marker

Contrast agent, e.g. MRIBiodegradable

polymer, e.g. polylactide

or

Release receptor, e.g. pH-, T-, UV-sensitive

Cell receptor

Other receptors, e.g. cell death receptor

Drug

Formulation of small and stable droplets by using high shear (e.g. ultrasound)Formulation of small and stable droplets by using high shear (e.g. ultrasound)

Principle of the miniemulsion process

high speedstirring,

ultrasound

Narrowly distributed nanodroplets Size range: 50-500 nmNarrowly distributed nanodroplets Size range: 50-500 nm

reaction

Reactions in confined geometries

1:1 copy (nanoreactor)

1:1 copy (nanoreactor)

Phase I

Phase II

diffusion of oil through the water

phase

Ostwald ripening: +

collision and fusionof

oil droplets

Coalescence:

Growth of droplets

Suppression of Ostwald ripening:

Addition of a co-stabilizer with low solubility in a continuous phase

Force: Same chemical potential in each dropletForce: Same chemical potential in each dropletK. Landfester, Macromol. Symp. 2000, 150, 171-178.

Suppression of coalescence:

Effective surfactants

(CH2)11CH3 SO4- Na+

C16H35 OCH2CH2 OH50

Lutensol AT50

Sodium dodecylsulfate (SDS)

Direct and Inverse miniemulsions

non-polar phaseand hydrophobe

H2O

surfactant surfactant

cyclohexane

polar phaseand lipophobe (e.g. salt)

block copolymere.g. poly[(ethylene-co-butylene)-b-(ethylene oxide)]

e.g. sodium dodecylsulfate (SDS)cetyltrimethylammonium chloride(CTMA-Cl)

StyreneOil-soluble initiatorOil-soluble fluorescent dye

WaterSurfactant

Water-soluble comonomer: e.g.PEG-acrylate, vinyl phosphonicacid, aminoethyl methacrylate, etc.

Oil-soluble comonomer: e.g.acrylic acid, glycidyl meth-acrylate, etc.

Oil phase Aqueous phase

Polystyrene functionalized nanoparticles

H3N+

NH3+

NH3+

NH3+

NH3+

NH3+

NH3+ NH3

+OH

OH

OH

OH

OH OHOH

OO

O

O

O

O

COO

COO

COOCOO

COO

COO

COO

COO

PO3 2

PO32

PO32

PO3 2

PO3 2

PO32

Langmuir, 2007, 23(10), 5367-5376. Colloid Polym. Sci., 2009, (in press).

StyreneOil-soluble initiatorOil-soluble fluorescent dye

WaterSurfactant

Water-soluble comonomer: e.g.PEG-acrylate, vinyl phosphonicacid, aminoethyl methacrylate, etc.

Oil-soluble comonomer: e.g.acrylic acid, glycidyl meth-acrylate, etc.

Oil phase Aqueous phase

Polystyrene functionalized nanoparticles

H3N+

NH3+

NH3+

NH3+

NH3+

NH3+

NH3+ NH3

+OH

OH

OH

OH

OH OHOH

OO

O

O

O

O

COO

COO

COOCOO

COO

COO

COO

COO

PO3 2

PO32

PO32

PO3 2

PO3 2

PO32

Dispersion of magnetite

J. Phys.Condens. Mat. 2003, 15, S1345-1362.

Encapsulation of materials in nanoparticles

One colloid particle per polymer particle:CaCO3 in PS

Macromol. Symp. 2000, 151, 549.

250 nm

Macromol. Chem. Phys. 2003, 204, 22.

Many colloid particles per polymer particle:Fe3O4 in PS

100nm

50 nm

One aggregate per polymer particle:Carbon black in PS

Macromol. Chem. Phys. 2001, 202, 51-60.

Particle size and surface groups density can be adjusted by varying the type and amount of surfactant/functional monomer Particle size and surface groups density can be adjusted by varying the type and amount of surfactant/functional monomer

Characterization of functionalized nanoparticles

Langmuir, 2007, 23(10), 5367-5376.

Poly(styrene-co-acrylic acid)

1 µm1 µm2 wt%, Dz=165 nm 0.5 wt%, Dz=220 nm,

1 µm

0.5 wt%, Dz=170 nm

400 mg Lutensol AT50

200 mg Lutensol AT50

0 wt% NH3+

Biomaterials, 2006, 27(14), 2820-2828.

Increase of surface functional groups amountIncrease of surface functional groups amount

0

5

10

15

20

25

30

nFL1

COO-

NH3+

Particle - Cell interaction

Surface functional groups density influence the cellular uptake Surface functional groups density influence the cellular uptake

3 wt% NH3+

HeLa cellsHeLa cells

20 wt% NH3+15 wt% NH3

+

b a

d c

20 μm 20 μm

20 μm 50 μm

Musyanovych A., et al. In „Clinical Chemistry Research“, Mitchem, B. H. and Sharnham, C. L. (ed.); Nova Science Publishers, Inc., 2009, Chapter VI.

H3N+

NH3+

NH3+

NH3+

NH3+COO

COO

COO

COO

COO

COO

COO

CO

O

Functional building block 1(TNF nanocyte)

Functional building block 2(Lipid layer)

Funktional building block 3(PEG-scFv = Ligand)

Funktional building block 4(cleavable PEG chain)

Bioactive multifunctional composite particles

Fluorescent aminefunctionalizedparticle

J. Control. Release 2009, 137, 69-77.

Induced drug release of the tumor necrosis factor TNFInduced drug release of the tumor necrosis factor TNF

Bioactive multifunctional composite particles

100 nm

Cryo-TEM

free lipids/liposomes

non-encapsulated particleslipid-encapsulated particles

- specific - non-specific

FACS

H3N+

NH3+

NH3+

NH3+

NH3+

J. Control. Release 2009, 137, 69-77.

cleavage in cellby enzyme

tumor necrosis factor becomes active

Bioactive multifunctional composite particles

Solvent

WaterPoly(L-lactide)

Poly(ε-caprolactone)

Poly(lactide-co-glycolide)Hydrophobic compound, e.g. marker, drug, etc.

Polymer precipitation within a nanodroplet

WaterSolvent evaporation

Macromol. Biosci., 2008, 23(10), 5367-5376.

Polymer

Particle size and size distribution mainly depend on the amount and type of polymer used Particle size and size distribution mainly depend on the amount and type of polymer used

Solvent

Water

Magnetite

WaterSolvent evaporation

Polymer

Biodegradable magnetite particles

Effect of magnetite amountEffect of magnetite amount

6.7 wt% 20 wt% 50 wt%

Macromol. Chem. Phys. 2009, 210, 961.

The rate of polymer degradation mainly depends on the type of surfactant, molecular weight and Tg of polymerThe rate of polymer degradation mainly depends on the type of surfactant, molecular weight and Tg of polymer

Degradation of nanoparticles

TEM

Release of magnetite from poly(L-lactide)

particles (MSC)

Release of magnetite from poly(L-lactide)

particles (MSC)

Release of fluorescent dye from poly(L-lactide) particles (HeLa cells)

Release of fluorescent dye from poly(L-lactide) particles (HeLa cells)

CLSM

Macromol. Biosci., 2008, 23(10), 5367-5376.

Crystallization in Gelatin Microgels

Gelatine in water droplets

X-linkingTransfer to H2O

Loading withCaCl2+ crystallizationby Na2HPO4

ApatiteCa10(PO4)6(OH)2 in gelatin microgels

Particle size: 220 nmCross linking with glutaraldehyde

Biomacromolecules, 2008, 9(9), 2383. Adv. Funct. Mater., 2008.

Encapsulation of liquids in miniemulsion

Polymerizationand phase separation

polymerhydrophobic oil

500 nm

Capsules via phase separation

Demixing

Styrene/ Hexadecane

Langmuir 2001, 17, 908-917.

Final morphology depends on:

• the interfacial tension between threedifferent phases (polymer, continuousphase, dispersed nanodroplet)

• kinetics of the polymerization vsphase separation

• miscibility of the phases

PMMA/Parfume

Macromol. Chem. Phys. 2009, 210.

Capsules via polymer nanoprecipitation

Water

Solvent/Non-solventSolvent evaporation

Water

Non-solvent

200 nm

poly(L-lactide) capsulespoly(L-lactide) capsules

in inverse miniemulsion

200 nm

poly(ε-caprolactone) capsulespoly(ε-caprolactone) capsules

Macromol. Biosci. 2006, 6(1), 33-40

Polymer

Capsules via reaction at the interface

Oil

Water, hydrophilic compounds, e.g. salt, contrast agent, DNA, etc.

Polymeric shell

+

Polyurea

HO OHR

O=C=N N=C=O

R' C

O

C

O

NH

R NH

NH

R' NH

C

O n

C

O

C

O

OR O N

H

R' NH

C

O nH2N NH2R

or or

Polyurethane

Oil

M1

Hydrophilic monomer

Addition of M2Crosslinking reaction at the interface

Redispersion in aqueous phase

Water

Crosslinking:M1:

M2:

Capsules via reaction at interface

+

Polyurea

HO OHR

O=C=N N=C=O

R' C

O

C

O

NH

R NH

NH

R' NH

C

O n

C

O

C

O

OR O N

H

R' NH

C

O n

C

O

NH

R'N=C=O

n

+ OH2 CO2C

O

NH

R'NH-COOH

C

O

NH

R'NH2 +

n

H2N NH2R

n

or or

Polyurethane

Crosslinking:

Hydrolysis:

Polyurethane Polyurea Crosslinked starch

M1:

M2:

200 nm

Langmuir, 2009, (in press).Macromolecules 2007, 40, 3122.

Capsules via reaction at interface

CH2 C

n

HO

CN

O=C

C4H9

O

CH2 C

CN

O=C

C4H9

O

-

-1

H+CH2 C

n

HO

CN

O=C

C4H9

O

CH2 CH

CN

O=C

C4H9

O

-1

Anionic polymerization of n-butylcyanoacrylate (BCA)Anionic polymerization of n-butylcyanoacrylate (BCA)

Oil

dsDNA(790 bp)

PBCA shell

CH2 C

CN

O=C

C4H9

O

OH-

n

1000

600

400

200

100

DNA- Marker Amount of dsDNAinside the droplets

Amount of dsDNAinside the PBCA capsules

About 15% of encapsulated DNA is in a form of free chainsProgr. Colloid Polym. Sci., 2008, 134, 120.

Increase of n-butylcyanoacrylate concentrationIncrease of n-butylcyanoacrylate concentration

Capsules via reaction at interface

Progr. Colloid Polym. Sci., 2008, 134, 120.

50 nm50 nm

200 nm200 nm

Reaction in the nanocapsule, e.g. reduction of Ag+ to Ag

Capsules as nanocontainers

Macromolecules 2007, 40, 3122-3135.

OH

H

OH

H

OH

H

O

H H

O

H H

O

H H

OH

H

OH

H

OH

HGd3+

Gd3+

Gd3+ Gd3+

Hydrophilic Gd complexes for magneticresonance imaging (MRI)Biomedical application

Macromol. Chem. Phys. 2007, 208, 2229-2241.

PCR

Multiplication of dsDNAinside the drolpets

Biomacromolecules 2005, 6(4), 1824-1828

Droplets as “Bioreactors”

Oil + SurfactantdsDNA

Primers

dNTPs

Taq DNA Polymerase

Taq 10x BufferMechanical

stirring

UltrasonicationPCR,

several cycles

Biomacromolecules 2005, 6(4), 1824-1828

Polymerase Chain ReactionPolymerase Chain Reaction

DNA- Marker

1500 bp

600 bp

400 bp

200 bp

100 bp

DNA template: 286 bpPCR – product: 135 bpDNA template: 286 bpPCR – product: 135 bp

CLSM TEM

Release of magnetite from poly(L-lactide)

particles in MSC

Release of magnetite from poly(L-lactide)

particles in MSC

Polyurea capsules with fluorescein

taken up by HeLa cells

Polyurea capsules with fluorescein

taken up by HeLa cells

Crosslinked starch capsules with rhodaminetaken up by HeLa cells

Crosslinked starch capsules with rhodaminetaken up by HeLa cells

CLSM

Cellular uptake

Langmuir, 2009, (in press).

Release mechanisms

1. Slow release• Diffusion from the nanocapsules:

release depends on the shell thickness and type of polymer

• Degradation of the nanocapsules:release depends on (bio)degradibility

2. Fast release•„switch“ e.g. by temperature, pHchange, enzyme…

Incorporation of blasting agent inside the capsule

Macromol. Mater. Eng. 2007, 292, 1237-1244.

without azo compound

with azo compound

Hydrophilic surface

uptake by cells

Inert polymer, e.g. polystyrene

Biodegradable polymer, e.g. polylactide

or

Cell receptor

Other receptors, e.g. TNF

specifity to certaincells

Summary

Release receptor, e.g. pH, T, UV-sensitive, enzyme…

defined release

“Stealthness”, e.g. PEG-chains

stable in blood stream

Fluorescent marker

Contrast agent, e.g. MRI

D marker included

encapsulation of drugs

Drug

Grit Baier(Cross-linked starch and PBCA capsules, PCR)

Eva Rosenbauer(Polyurea capsules)

Markus Urban(Polylactide composite particles)

Anika Hamberger(PMMA capsules with blasting agents)

University of UlmProf. Paul WaltherDr. Oliver ZimmermannDr. Juliane Weich

Institute of Cell biology and Immunology, University of Stuttgart

Prof. Roland KontermannSylvia Messerschmidt

Acknowledgments

MPI for Polymer Research

Dr. Anitha EthirajanDr. Daniel CrespyDr. Ingo Lieberwirth

Dr. Umaporn Paiphansiri(Polyurethane capsules)

DFG (SPP1259, 1166, SFB); Max Planck Society; EUVW-Stiftung; Fonds der Chemischen Industrie; Degussa/EvonikLandesstiftung BW; BASF; Bayer Materials; Clariant

Financial support: