1

Improved alginates for cellencapsulation by the use ofenzymatic engineering

Berit Løkensgard Strand, Ph.D.Neuchatel July 6th, 2005

Neuchatel July 6, 2005

Microcapsules in cell therapy

(Lanza et al. 1999)

Immune cells

Immunoisolation:

Cells Capsule

Nutrientsand oxygen

Cellproducts

Insulin

Graft rejection +autoimmunedisease

Cell

Insulin

Oxygen andnutrients

Waste products

Capsulemembrane

Cytokines, free radicalsreactive oxygen- and nitrogen-

intermediates

Antigen-presenting cells

Secreted proteins

LymfokinesCytotoxic cells

T-cells

B-cells Antibody-producing cells

Macrophages

Antibodies

Autoimmuneantibodies

Complementcomponents

Y

Naturaloccurringantibodies

YY YYY

cv YYv c

Y cc

Cellular response Humoral response

Fibroblast

Encapsulation procedure – thestarting point

• For non-proliferating tissue or cells,a mild encapsulation procedure isneeded (e.g. Pancreatic islets,stem cells).

• For proliferating tissue, a mildencapsulation procedure is not thatimportant since dead cells can bereplaced by new living cells

History of microencapsulation (EC)

• 1964: First description ofmicroencapsulation by T.M.S. Chang.

• 1980: Microencapsulated islets asbioartificial pancreas by F. Lim and A.M. Sun.

In vivo (syngraft):Transplanted ECislets are able toreversehyperglycemia indiabetic animals

In vitro:Encapsulated isletsfunction as non-encapsulatedislets

Lim and Sun, Science 1980

History of microencapsulationin transplantation

Since then (1980-2005):Capsule characterisation (and improvements):

- stability- permeability- biocompatibility

Technical improvements:- Bead generators for formation of small andevenly sized beads

- Purification of materialsTransplantation:

- Allo- and xenotransplantation- Larger animal models- Clinical trials

Problem:- Promising highlights but problems

with reproducing results

Important Capsule Properties

• Stability• Permeability• Size• Biocompatibility

CaCl2

AlginateCells

Formation of Ca-alginate gel beads

Alginate

GM M M MGGGGGGGMGMGMGMGM M M M M MG

M - block G - block MG - block M - block

HH

OH

OH

OH

OH

OHH

COO-

H

-D-Mannuronic acid (M)

HH

OH

H

OHCOO-OH

OH

OHH

-L-Guluronic acid (G)

O

OO O

OO

O

O

O

O

COO- COO-

COO-

OH

OHOH

OH

HO

OH HO

COO-

-OOC

OH

OH

OH

G G GM M

G : 1C4

M : 4C1

Alginate properties depend onalginate composition

Ca2+

Ca2+

O

O

O

OH

OHO

OH

¯OOC

¯OOC

OH

G G

• Alginates gel forming properties with divalent cationsdepend on the G-content as the G-blocks specificallybinds the divalent ions:

Alginate sourcesand composition

Alginate source FG FM FGG FMMFGMFMG

FGGG FGGM FMGM NG>1

Durvillea antarctica 0.32 0.68 0.16 0.51 0.17 0.11 0.05 0.12 4Macrocystis pyrifera 0.42 0.58 0.20 0.37 0.21 0.16 0.04 0.17 6

Laminaria hyperborea, leaf 0.49 0.51 0.31 0.32 0.19 0.25 0.05 0.13 8

L. hyperborea, stipe 0.63 0.37 0.52 0.26 0.11 0.48 0.05 0.07 15

Pseudomonas sp. 0 - 0,5 0

Azotobacter vinelandii 0,10-0,85 0,02-0,85

Algal alginates:

Bacterial alginates: FG FGG

L. hyperborea, outer cortex 0.71 0.29 0.57 0.16 0.13 0.54 0.03 0.10 20

Important Capsule Properties

• Stability• Permeability• Size• Biocompatibility

CaCl2

AlginateCells

Formation of Ca-alginate gel beads

• Stability• Permeability• Size• Biocompatibility

• Gelling ions – type and concentration• Alginate concentration,composition and MW (< 2-3x105)

• Distribution of alginate in the capsule• Adding a polycation layer• Size

What DeterminesImportant Capsule Properties

Stability in saline solution- Swelling of alginate beads

50mM CaCl2 50mM CaCl2 +1mM BaCl2

10mM BaCl2 50mM SrCl2

Dia

met

er(µ

m)

Change of NaCl-solution

High-G alginate (69% G)

400

500

600

700

800

900

1000

0 1 2 3 4 5 6 7

High-M alginate (43% G)

400

500

600

700

800

900

1000

0 1 2 3 4 5 6 7

The alginate distribution in the geldepends on the gelling conditions

50mM CaCl2in 0.3M mannitol

0

50

100

150

200

250

0 100 200 300 400 500 600 700

Intensity profile

Distance (µm)

10mM BaCl2in 0.3M mannitol

Distance (µm)

0 100 200 300 400 500 6000

50

100

150

200

250Intensity profile

50mM SrCl2in 0.3M mannitol

Distance (µm)

Intensity profile

0

50

100

150

200

250

0 100 200 300 400 500 600 700

Addition of polycation increases capsulestability and reduces capsule permeability

Polycation(Polylysine (PLL,PDL), chitosan, etc.)

Alginate

Ca-alginate gel beads

Alginate-polycation microcapsules

Reduction in size reduce capsulestabilityBecause more of the gel is exposed to the surface of a small gel than a largergel, smaller gels are more vulnerable to destabilisation than larger gels:

Mannitol wash,Exposure to 0.10% PLL

Saline wash,Exposure to 0.05% PLL

Destabilization upon PLL-exposure (200µm beads):

0 4 8 12 16Time of exposure to PLL (min)

0

20

40

60

80

100

Frac

tion

ofin

tact

caps

ules

(%)

Saline wash,Exposure to 0.10% PLL

What DeterminesImportant Capsule Properties• Stability

• Permeability• Size• Biocompatibility

• Alginate gel: 1-2% alginate (98 %water/buffer) gives high diffusionrates for small molecules such asoxygen and glucose

• Composition: High-G alginatesgels are more permeable thanhigh-M alginate gels

• Adding a polycation layerreduces the permeability of themicrocapsules

What DeterminesImportant Capsule Properties• Stability• Permeability

• Size• Biocompatibility

• Reduction in size increase thediffusion of oxygen and nutrientsto the EC cells

• New technology reduce the sizeof high viscous droplets, hencemake beads about 150-200 μmin diameter with a narrow sizedistribution

• Reduction in size decrease thecapsule stability

What DeterminesImportant Capsule Properties

• Stability• Permeability• Size

• Biocompatibility

• Alginate is a non-toxic polymer• In transplantation: Cells growing

on the capsule surface reducethe diffusion of nutrients andoxygen to the EC cells, and maysecrete products that harms theEC cells

• This fibrotic overgrowth may becaused by

• products secreted from the EC cells• the surgical procedure• endotoxins in the materials• the polycation

0.1%PLL exp.10minCapsules without fibrosis:

0%, n=6

0.05%PLL exp.5minCapsules without fibrosis:

91 ± 5%, n=3

without PLLCapsules without fibrosis:

91 ± 6%, n=3

Fibrosis on empty alginate-PLL-alginate capsulesis dependent on the poly-L-lysine (PLL) coating

Effects of PLL on tumor necrosis factor (TNF)production and necrosis in monocytes

10

100

1000

10000

20

40

60

80TNF

Necrosis

10 1000Concentration of PLL (g/ml)

TN

F(p

g/m

l)

%ne

cros

is

COMPOSITION+ SEQUENCE

SWELLING / SHRINKAGE

DIFFUSIONPROPERTIES

(porosity, charge)

MECHANICALPROPERTIES(gel strength)

CHEMICALSTABILITY

(towards ions andcalcium chelators)TRANSPARENCY

BIOLOGICALACTIVITY

CHARGE DENSITYbinding of polycation

Alginate properties depend onalginate composition

-D-ManpA

-L-GulpA

OHOOC

HO

OH

OH OHO

OHO

HOOCOHO

HOOCO

O O

OHO

HOOC

OH

O

Mannuronan C-5 Epimerase,(AlgE4)

HO OO

H

OHO C

HO OO

HOOC

OH

OHO

OH OHOOC

HO

O

HOOC

O

OH

OO

M M

M M M

GG

M

Epimerases catalyze the conversionof M to G in the alginate chain

AlgE1

AlgE2

AlgE3

AlgE4

AlgE5

AlgE6

AlgE7

G-blocks, MG-blocks

Short G-blocks

MG-blocks

G-blocks

Long G-blocks

Lyase activity+ G-blocks, MG-blocks

---

A - 385 amino acidsR - 155 amino acids

(Ertesvåg et.al., Glærum et.al.)

A1 R1

R4A1 R1 R2 R3

A2A1 R1 R2 R3 R4

R5 R6A2A1 R1 R2 R3 R4 R7

R4A1 R1 R2 R3

A1 R1 R2 R3

A1 R1 R2 R3

Epimerases Activities

-D-ManpA

-L-GulpA

OHOOC

HO

OH

OH OHO

OHO

HOOC

OHO

HOOCO

O O

OHO

HOOC

OH

O

C-5 Epimerase AlgE4

HO OO

H

O

HO C

HO O

O

HOOC

OH

OHO

OH OHOOC

HO

O

HOOC

O

OH

OO

M M

M M M

GG

M

0 100 200 300 400 500 600 700Distance (µm)

0

50

100

150

200

250

Intensity

Profile

Capsules of epimerised alginates are smallerAnd more inhomogeneous than capsules

from the original alginate sample

A B

0 100 200 300 400 500 600Distance (µm)

0

50

100

150

200

250

Intensity

Profile

d = 550 μmV = 0,09 mm2

d = 620 μmV = 0,12 mm2

Change of NaCl-solution

0

1

2

3

4

5

0 1 2 3 4 5 6 7

V/V

o

M.pyrifera M.pyrifera + AlgE4

400

500

600

700

800

0 1 2 3 4 5 6 7

Dia

met

er(µ

m)

Stability in saline solution- Swelling of alginate beads

0

0,2

0,4

0,6

0,8

1

1,2

0 20 40 60

Time [min] in water

Fra

ctio

no

fin

tact

caps

ule

s

0

20

40

60

80

100

120

140

160

0 20 40 60

%In

crea

seof

cap

sule

diam

eter

L.hyperborea stipe L.hyperborea stipe + AlgE4

Stability against osmotic pressure- Swelling in ion free water

0

500

1000

1500

2000

2500

Bou

nd

IgG

[cp

m]

Positive controlUnspesific bindingOriginal alginateEpimerised alginate

Controls M. pyrifera(43% G)

L. hyperborealeaf (52% G)

L. hyperboreastipe (65% G)

Ca/Ba-alginate beads are less permeable to IgGafter epimerisation with AlgE4

Permeability (IgG, 150 kDa)

05000

100001500020000

2500030000

Bo

un

dT

NF

[cp

m]

Positive controlUnspesific bindingOriginal alginateEpimerised alginate

Controls M. pyrifera43% G

L. hyperborealeaf (52% G)

L. hyperboreastipe (65% G)

Alginate-PLL-alginate capsules are less permeableto TNF after epimerisation

Permeability (TNF, 55 kDa)

0

20

40

60

80

100

0 0,05 0,1

Conc. of polycation (%)

%b

indi

ng

(TN

F) High-G, PLL

High-G + AlgE4, PLL

High-G, PDL

High-G + AlgE4, PDL

Porosity of alginate-polylysine-alginateCapsules to TNF

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 20 40 60

Time [min] in water

Fra

ctio

no

fin

tact

cap

sule

s

0,035%PDL

0,05%PDL

0,05%PLL

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 20 40 60

High-G: High-G + AlgE4:

Stability against osmotic pressure- Swelling in ion free water

MG coating-alginate binds better to the alginate-PLLcapsule than block alginate and poly-M

0

50

100

150

200

250

0 2 8 15 21

Time of storage (days)

µg

coat

ing

algi

nate

/mlc

apsu

les

Block-alginate:FG = 0.45

poly-M:FG = 0.06

poly-MG:FG = 0.41

Coating the PLL-layer with poly-MG alginatereduces the overgrowth on implanted empty capsules

Ret

rieva

l(%

)

0

10

20

30

40

50

60

70

Standard Poly-MGcoating

High-G High-G(epim.)

0

50

100

150

200

250

300

350

Standard Poly-MGcoating

High-G High-G(epim.)

Glu

cose

oxid

atio

n(p

mol

/10

caps

ules

x90m

in)

Rejection of encapsulated graft

Exposedpolycation

Mechanicalfailure

Insufficientimmune protection:• Protruding cells• Permeablemembrane

Alginate

Alginate gel PolycationCells

Leaking materials:- cell products- PLL- high M-alginate- impurities

CONCLUSION (1)

Smaller capsules Reduced porosityIncreased resistance to swelling Stronger gel

By epimerising the core alginate we are able to reducethe toxic PLL layer and still keep the stabile and immune

protective behavior of the capsule

Epimerisation of the core alginate with the alternaseAlgE4 gives:

CONCLUSION (2)

Epimerisation of the coating alginate with the alternaseAlgE4 gives:

Better binding to the PLL-layer

By epimerising the coating alginate we are able to coatthe toxic PLL layer better and thus increase the

biocompatibility of the capsules

Reduced overgrowth on implanted empty capsules

Department of Biotechnology:Gudmund Skjåk-Bræk (Prof)Berit Løkensgard Strand (PhD)Ivan Donati (PhD) (University of Trieste)Yrr A. Mørch (PhD student)Wenche Strand (Bioengineer)Sissel Tove Ødegaard (Bioengineer)

Department of Cancer Research and Molecular Medicine:Terje Espevik (Prof)Bård Kulseng (MD)Anne Mari Rokstad (PhD student)Kristin Rian (MSc)Liv Ryan (Bioengineer)Bjørg Steinkjer (Bioengineer)

NTNU:

THE TRONDHEIM BIOENCAPSULATIONGROUP

www.alginatecapsules.com

Department Cell Biology:Arne Andersson (Prof)Stellan Sandler (Prof)

University of Uppsala, Sweden:

International Collaboration

Department Surgery:Ray Rajotte (Prof)Greg Korbutt (Ass. Prof)

University of Atlanta, Canada:

Igor Lacik (Prof, Polymer Institute of the Slovak Academy of Sciences)Dr. David Hunkeler (AQUA+TECH Specialties S.A.)