Intelligent Micro- and Nanocapsules

Gleb SukhorukovMax-Planck Institute of Colloids and Interfaces,

Golm/Potsdam, Germany

Coating colloids and Hollow capsulesRelease properties

Encapsulation of macromoleculesOrganic dye precipitation by pH-gradientInorganic particles synthesis in capsules

Poor-water soluble dye precipitationCapsule based combinatorial libraries

Enzymatic reaction in capsules

Polyelectrolyte Layer-by-layer assembly

3. Polycation Adsorption

4. Wash

1. Polyanion Adsorption

2. Wash

Capsule preparation

Hollow Polyelectrolyte Capsule

Organic and inorganic colloidal particles,

drug nanocrystals, biological cellsCores

The core (template) is a dissolvable colloidal particle, a drug particle a dye particle or

even a biological cell

Melamin resin coresMelamin resin coresInorganic cores, carbonates, oxidesInorganic cores, carbonates, oxidesDye and drug particles Dye and drug particles DropletsDropletsErythrocytes, others biological cellsErythrocytes, others biological cells

Layer constituents

Synthetic polyelectrolytesSynthetic polyelectrolytesBiopolymers (proteins, polysacharides, nucleic Biopolymers (proteins, polysacharides, nucleic acids)acids)Lipids,Lipids,Inorganic nanoparticlesInorganic nanoparticlesThe wall can be tuned in thickness, composition The wall can be tuned in thickness, composition and functionality by choosing various constituents and functionality by choosing various constituents and adjusting the layer numberand adjusting the layer number

Advantage of the technique

The size and shape of the The size and shape of the capsules is controlled by the capsules is controlled by the SIZE and SHAPE of the SIZE and SHAPE of the TEMPLATETEMPLATE

Rather monodisperse capsule Rather monodisperse capsule dispersions can be prepared

Templated on red blood cell

dispersions can be prepared

Templated on melamin particles

0 1 2 3-80

-60

-40

-20

0

20

40

60

Fluorescently labeled polyallylamine concentration (microgram/ml)

0

5000

10000

15000

20000 supernatant flu

orecenceZe

ta-p

otentia

l

( )

NH3+

Poly(allylamine hydrochlorid) PAH

C l-

Poly(styrenesulfonat) PSS

( )

SO3-

N a +

Next layerMatched concentration

Next layerCentrifugation

Next layerFiltration

Next layerGel-electro-phoresis

Electrophoretic Mobility measurements as means to follow Layer Growth

Layer Number0 2 4 6 8

-80

-60

-40

-20

0

20

40

60

ζ - P

oten

tial [

mV

]

Various Polyelectrolytes: PDADMAC - PSS BSA - PDADMAC, PSS - PAH, DNA - PDADMAC

Layer Thickness - Monitoring of Multilayer Formationby Single Particle Light Scattering

200 300 400 500 600 7000

500

1000

1500

2000

21 layers11 layers

control

Scattering Intensity (arb. units)La

yer T

hick

ness

[nm

]

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16

Layer Number

Scanning electron microscopy Atomic force microscopy

Layer-by-Layer approach

Templating on biological cells - MICROREPLICA

Echinocyte cells

Confocal Scans through an Echinocyte templated polyelectrolyte shell

Release Control by Multilayers

0 100 200 300 400 500 600 700 800 900 1000 1100

0

1x104

2x104

3x104

4x104

5x104

Uncoated Fluorescein particles 9 PSS/PAH layers 13 PSS/PAH layers 15 PSS/PAH layers 18 PSS/PAH layersFl

uore

scen

ce, a

.u.

Time, sec

0 2 4 6 8 10 12 14 16 18 20

0

1

2

3

4

5

6

7

Perm

eabi

lity

coef

ficie

nt, m

/sec

*10-8

Number of layers

Permeability Coefficients: Ionic Strength

Ionic strength influencesdrastically (one order of

magnitude going from 1 to 500mM) the properties of

PEM.

0,0 0,1 0,2 0,3 0,4 0,50,0

0,5

1,0

1,5

2,0

2,5

3,0

Perm

eabi

lity

Coe

ffici

ent,

m/s

ec *1

0-8

Ionic strength, M

0 5 10 15 20 250

20

40

60

80

100

120

140

Fluo

resc

ence

, a.u

.

Time, min

0,0 0,5 1,0 1,5 2,0 2,5 3,00

20

40

60

80

100

120

140

1mM 10mM 25mM 50mM 100mM 250mM 500mM

Permeability behavior of annealed shells

20 30 40 50 60 70 80 90 1001E-9

1E-8

1E-7

1E-6

1E-5

Perm

eabi

lity

[m/s

]

Temperature [ °C]

Annealing time = 30 min

10 min

Capsules 8 layers PAH/PSS

Heat Treatment can close defects

The Presence of the Lipid Bilayer Decreases the Permeability

Capsules with 8

Polyelectrolyte LayersCapsules with a Phospholipid Bilayer

The fluorescent polar marker 6-carboxyfluorescein is excluded

Encapsulation via Permeability RegulationDextran-FITC

pH >7.5, closed state pH <6.5, open state pH>7.5 Encapsulated

1 2 3 4 5 6 7 8 9 10 11 12

open capsules

closed capsules

pH

Encapsulation of Enzymes

0

20

40

60

80

100

50210%

of a

ctiv

ity fr

om n

ativ

e[BPTI] / [α-chymotrypsin], mol/mol

free α-chymotrypsin encasulated α-chymotrypsin

0 1 2 3 4 5 6 7 80

20

40

60

80

100 pH8.0 , 25oC Native chymotrypsin Encapsulated chymotrypsin

Res

idua

l act

ivity

, % fr

om T

ime=

0

Time, days

Inhibitor

Enzyme

Encapsulation of Urease in Polyelectrolite Multilayer shells

Ethanol/Water 1:1In WaterIn Water.

Encapsulated

5 mµ5 mµ

Calcium carbonate growth into polyelectrolytecapsules by urease catalyzed reaction

A final stage of precipitation

Polymer Synthesis inside Capsules

pH = 1

Monomers

80°CS O2 8

2-

Polymerisation Washings

Broken and Empty Capsules

Swelling as a result of osmotic pressure increase.•The capsules were filled with a fluorescent

(rhodamin) copolymer

•The permeability difference between substrate and product is always employed

Encapsulation of macromolecules. 1. Controlled precipitation of polymers on colloidal particles

Precipitating condition

Solvent or complex-ion

10µm

Encapsulation of macromolecules. 2. Inner shell decomposition

non-chargedpolyanion Me3+

charged

Reversible shrinkage - swelling of the loaded with polyelectrolyte (PSS) capsules induced by osmotic

pressure

Reversabl e shrinkage - swelling of the loaded with polyelectrolyte (PSS) capsules induced by osmotic pressure

681012

Diameterofthecapsules(µm)

0 1 2

6

8

10

12

Dia

met

er o

f the

cap

sule

s (µ

m)

PSS concentration in bulk solution (monoM)

Organic dye precipitation by pH-gradient

Electroneutrality (C = C )yields pH = 7

OH H

neutralacid

H+

pH- difference through capsule wall established by Donnan equilibrium

Polycationpolyanion

neutral

basic

neutral

acid

acid

neutral

basic

neutral

Organic dye precipitation by pH-gradient

Polyelectrolyte Shells as templatesfor controlled crystallization and

precipitation of small organic molecules. Model of drug loading pH-difference

Fluorescence Confocal Image (self-quenching) Transmission Image

Scanning electron microscopy image of carboxytetra-methylrhodamine precipitates 6-carboxy-fluorescein

Organic dye precipitation inside capsules caused by pH-gradient

solubility, nucleus sites, pH-difference

6-CF 5(6)-TAMRAM X U EI T R

Inorganic particles synthesis inside capsules

pH-difference

Selective pH-induced formation of Iron oxide crystals into capsule

filled with polycation

23

Optical microscopy

TEM

Hematite Fe2O3-particles

Magnetite particles synthesis in capsule interior

Fe3+Fe2+

pH-difference

OH- Fe3O4

Selective polymer/light-induced Silver particles formation into PSS and dextran filled capsules

Light

Ag+

Optical microscopy image of dextran filled capsules

in AgNO3 solution

TEM image of dextran filled capsules in AgNO3 solution

Precipitation poor-water soluble dyes in capsules caused by polarity gradient

Filled capsules

Suspension of capluces with precipitated drug in water

Capsules+drug suspension in acetone/water mixture (high acetone content)

DR-1

0,0 0,3 0,6 0,9

0,0

0,3

0,6

0,9

Acet

on c

once

ntra

tion

insi

de th

e sh

ells

Aceton concentration in outer solution

LbL of Thermosensitive Polyelectrolytes

use of charged PNIPAM derivativesfor the preparation of thermosensitive microcapsules

anionicblock copolymer(SS24-NIPAM76)

cationicblock copolymer

(DEAEMA34-NIPAM66)

C=O

NH

H3C CH3

66

C2H5

C=O(CH2)2

H5C2

34

NH+, Cl-

H3C

C=O

NH

H3C CH3

7624

, Na+SO3-

LCST = 31.9ºC LCST = 31.8ºC(determined by DSC measurements)

Influence of the Temperature on the Capsule

size measured by confocal microscopy (8 deposited layers)

-the thickness of the wall measured by AFM increases with the temperature-Ratio of thickness ≈ 0.5

⇒ decrease of the permeability of the wall

R

e << R ⇓

V = 4πeR2

volume Vat 25°C

x

increaseof the

temperature

e’ volume V'at 60°C

R’x

e’ << R’ ⇓

V = 4πe’R’2

V = V’ ⇒ (e/e’) = (R’/R)22.2

2.0

1.8

1.6

1.4

Radi

us (µ

m)

6050403020100Temperature (ºC)

R25

R’60

shrinking of the hollow capsules with the increase of the temperature

ratio R’60/R25 = 0.77

Combinatorial library based on Doping of Capsules with Fluorescent nanoparticles (quantum dots) and their mixture

500 600 700

1:1PL

inte

nsity

[a.u

.]

500 600 700

1:2

Wavelength [nm]500 600 700

2:1

Combinatorial library based on particle signing

Reduction of Ag in film by laser beam on surface of colloidal particles

Ag/PSS film was assembled on colloid particles

5 µm

Enzymatic reactions inside capsule

Ca2+ Chymotrypsine

Dextran sulfate/ protamine capsules filled with alginate

5 µΜ

Chymotrypsine embedding in capsules containing alginate gel

Enzymatic reactions inside capsule

Kinetic scheeme of chymotrypsime function

C hym o tryp sin

NH2

COO-

Rhodamine 110

Exc ita tio n - 498 nm

Em issio n - 521 nm

Enzymatic reactions inside capsule

Capsules with embedded chymotrypsine

Bi-enzyme system incorporated in the capsule

Pe ro xid a se fluo re sc e in la b e lle d

10µΜ

G luc o se o xid a se

Pe ro xid a se

Re d

Exita tio n 563 nme m issio n 587 nm

A m p le x fo r g luc o se a ssa y, c o lo rle ss

G luc o se o xid a se rho d a m ine la b e lle d

10µ 10µΜ Μ

Shell with defined Size, thickness, stability,

composition,Affinity properties

Controlling Uptake and release

Drug formulationDrug carriers

Sustained releaseTargeting

Microreactor,CatalysisSensing

MacromoleculeEncapsulation

Hollow particle

Precipitation,Product collection in capsule interior

Colloidal particle

Biological Functions onBiological Functions on PolyelectrolytePolyelectrolyte Capsules Capsules --TowardToward ArtificialArtificial CellsCells?

Lipid-Polyelectrolyte Interaction

Tuning the Lipidproperties Tuning of the polyelectrolyte

Layer properties

Do the membrane proteins work?

Additional Functions: Nanoparticles, Fluorescent

Reporter Molecules, Magnetic Particles, etc.

Synthese

Capsule or Colloidal Core

Acknowledgments.

Prof.Dr.Helmuth Möhwald Collaborators:Prof.Edwin Donath

Prof.Yuri Lvov,Prof.Olga Vinogradova

Prof.Natalia I. Larionova,Dr.Andrei Rogach

Dr.Alexander Petrov,Dr.Dmitry Shchukin,

Yuri Fedutik Michelle Prevot

Capsulution NanoScience AG

Postdoctoral researchers:Dr.Karine Glinel,Dr.Olga Tiourina,

Dr.Dinesh Shenoy,Dr. Claire Peyratout

Dr.Radostina Georgieva

Ph.D.Students:Alexei Antipov,

Igor Radtchenko,Ana Cordeiro,Wenfei DongAnja Günther,

Technical staff: Carola Gaudl, Anne Heilig, Heidi Zastrow

Financial supportSofia Kovalevskaya Programm of Alexander von Humboldt Foundation,

BMBF- Investment for Future Programm, EU-project „Nanocapsules“