Encapsulation with Alginates

Berit L. Strand, Yrr Mørch and Gudmund Skjåk-BrækNorwegian University of Science and Technology

(Lanza et al. 1999)

Microcapsules in cell therapy

Endostatic producing cells for hindering re-growth of tumour tissue(gliomas)

Chondrocytes for the formationof new cartilage

Stem cells for the formationof new tissue

(Lanza et al. 1999)

Cells(Islets) Capsule

Nutrients and oxygen

Cell products

Immune cells

Immunoisolation:

Insulin

Graft rejection +autoimmune disease

Microcapsules in cell therapy

(chicagodiabetesproject.org)

Cell

Insulin

Oxygen and nutrients 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

(Adapted and modified from Colton 1996)

Important capsule parameters

• Stability• Biocompatibility• Permeability• Size

• Cell function after encapsulation

Biocompatibility

• Host immune cells may be stimulated by capsule components to secrete elements that are harmful to the encapsulated cells

• Host cells growing on the capsule surface reduces theamount of nutrients and oxygen that passes through thecapsule to the encapsulated cells

Biocompatibility - depending on exposureof poly-L-lysine (PLL)

0.1% PLL exp.10minCapsules without fibrosis:

0%, n=6 mice

0.05% PLL exp.5minCapsules without fibrosis:

91 ± 5%, n=3 mice

without PLLCapsules without fibrosis:

91 ± 6%, n=3 mice

Empty capsules transplanted to Balb/c mice and explanted after 4 weeks

(Strand et al. 2001)

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)

TNF

(pg/

ml)

% n

ecro

sis

(Strand et al. 2001)

Need of new solutions of controlling stabilityand permeability when omitting the polycation

Strategies• Using gelling ions of higher affinity to

alginate• Using high-G alginate with a high MW• Formation of inhomogeneous beads• Using enzymatically tailored alginates

(epimerased alginates)• Using chemoenzymatic strategy to

covalently crosslink the alginate

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

OO

O

O

COO- COO-

COO-

OH

OHOH

OH

HO

OH HO

COO-

-OOC

OH

OH

OH

G G GM M

G : 1C4

M : 4C1

(Fisher and Dörfel 1955; Atkins et al. 1970; Haug et al 1964-1967)

CaCl2 (or BaCl2 )

AlginateCells

Formation of Ca-alginate gel beads

(Smidsrød and Skjåk-Bræk 1990)

Alginate sources and composition

Alginate source FG FM FGG FMM FGMFMG 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 aeruginosa 0 - 0,5 0

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

Algal alginates:

Bacterial alginates: FG FGG

(Smidsrød and Skjåk-Bræk 1990)

Alginate properties depend on alginate composition

Ca2+

Ca2+

O

O

O

OH

OHO

OH

¯OOC

¯OOC

OH

G G

(Grant et al 1973; Smidsrød 1974)

Alginate properties depending on composition

GG/GG junctions

MG/MG junctions

GG/MG junctions

(Donati et al, 2005)

Alginate properties depending on alginate composition - stability

Dia

met

er (µ

m)

50mM CaCl2 50mM CaCl2 +1mM BaCl2

10mM BaCl2 50mM SrCl2

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

(Mørch et al. 2006)

Epimerisation of alginate with mannuronan C-5 epimerases

OOO

OHO OHO

-OOCHO

HO-OOC

O

OO

OOH

HO

OH

-OOC

-OOCOH O

OO

OOH

HO

OH

-OOC

-OOCHO O

OO

OOH

HO

OH

-OOC

-OOCHO

M M MM M M M M

-OOC

OO

-OOC

OH

OH

AlgE4

OO

OHO OHO

-OOCOH

OHO

-OOC

OO

OH

OHO

O

OHO OHO

-OOCOH

OHO

-OOCO

O

-OOC

OH

OH

OO

-OOC

OH

OH

M M GG M M GG

AlgE1/AlgE6

O O

OH

O

- OOC

HOO

-OOC

OH

OHOH

OHO

-OOC

OO

-OOC

OH

OH

O

OH

O

- OOC

HOO

-OOC

OH

OH

OO

OHO OHO

-OOC

OHO

HO

-OOC

OO

-OOC

OH

OH

OO

M M GG G G GG M

(Adapted and modified from Ertesvåg, Valla and Skjåk-Bræk 1996)

Change of NaCl-solution

Dia

met

er [

µm

]

Epimerised polyMG alginate (67% G)

High G alginate (69% G)

Epimerised polyMG alginate (56% G)

Epimerised High M alg (56% G)

Swelling of alginate beads- effect of epimerisation

400

500

600

700

800

900

1000

0 1 2 3 4 5 6 7 8

(Mørch et al. 2007)

Inhomogeneous alginate distribution

An inhomogeneous alginate core • (binds more PLL)• forms a more stable capsule• forms a less permeable capsule• (reduces the risk of protruding islets?)

0

50

100

150

200

250Intensity Profile

0 100 200 300 400 500 600

Microcapsule center

Permeability assay

Dynabeadcoupled withmice antibody(with anti-TNF specificity)

(Kulseng et.al.1997)

125-I labelledanti-mice IgG(or TNF)

Capsulemembrane

Radius of

gyration

(RG

) for a globular

molecule

is proportional to the

cubic

root

of

the

molecular

weight(a):

RG

Mw⅓

Insulin

5.8 kDa

TNF-α

51 kDa

IgG

150 kDa

Transferrin

80 kDa

IL-1β

17.5 kDa

(a Tanford, C. Physical chemistry of macromolecules. New York, John Wiley & Sons 1961)

Permeability of IgG

into beads of high-G alginate

0

1000

2000

3000

4000

5000

6000

7000

8000

Positive control Negative control Ca supplemented with Ba

cpm

Permeability (IgG, 150 kDa)

(Mørch et al. 2006)

Alginate properties depending on alginate composition - permeability

(Mørch et al. 2007)

0

10

20

30

40

50

60

70

80

90

100

Nega

tive c

ontro

lPo

sitive

contr

ol

L.hyp

, FG

= 0.65

M.py

r, FG

= 0.42

FG=0

.52FG

=0.55

FG=0

.61FG

=0.65

Bin

ding

of I

gG[%

]

Epimerised alginates

Native alginates

TNF55kDa

IgG150kDa

Capsules of different permeability

Alg/PDL/pMG Alg/PLL/pMG Alg

(Strand et al. 2003)

Human pancreatic isletsencapsulated in alginate microbeads

Day 1:

Day 16:

Encapsulated humanIslet in culture for 141 days:

(Strand 2006)

50

60

70

80

90

100

50

60

70

80

90

100

Viab

ility

(%)

Groups

non-encapsulated islets

encapsulated islets

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Stim

ulat

ion

inde

x (S

I)

Groups

non-encapsulated islets

encapsulated islets

Human pancreatic islets from Chicago encapsulated in alginate beads - viability and function after 2x overseas shipments and encapsulation

(Qi, Strand et al. 2008)

0 5 16 27 35 52 69 95 109 130 173 2080

100

200

300

400

500

600

non-

fast

ing

bloo

d gl

ucos

e (m

g/dl

)

0 5 16 27 35 52 69 95 109 130 173 2080

100

200

300

400

500

600

days (posttransplantation)

0 5 16 27 35 52 69 95 109 130 173 2080

100

200

300

400

500

600

0 5 16 27 35 52 69 95 109 130 173 2080

100

200

300

400

500

600

Encapsulated human islets to nude mice

10 000 IE 5000 IE

3000 IE 1000 IE

(Qi, Strand et al. 2008)

• Polycations provoke immune responses

• Alginate capsules without polycation can protect transplanted pancreatic islets in allo- and xenomodels (mice)

• Stable alginate capsules can be made by the right selection of alginate and gelling ions

• By enzymatic modification, new alginates can be made tailored for their use in encapsulation

Conclusions

“A group of highly qualified scientists and their teams who have committed themselves to achieving a functional cure for diabetesas soon as possible” (via transplantation of insulin producing cells)

• Finding new sources for insulin producing cells• Transplantation wihtout immuno suppression (encapsulation)

www.chicagodiabetesproject.org

Norwegian

University of

Science and TechnologyGudmund

Skjåk-BrækTerje

EspevikYrr

A. MørchAnne Mari RokstadLiv

RyanBjørg

SteinkjerWenche

Strand

Acknowledgements

The Chicago Diabetes Project, Encapsulation

Team:Chicago, Illinois, USA: Jose Oberholzer, Meirigeng

QiUrbana-Champagne, Illinois USA: Kevin Kim, Hyungsoo

ChoiBratislava, Slovakia: Igor LacikGeneva, Switzerland: David HunkelerSydney, Australia: Bernie Tuch

Financial Support:Norwegian

Research CouncilThe Norwegian

Diabetes Association via Extra

Funds from the Norwegian

Foundation for Health and RehabilitationThe Chicago Diabetes Project

University of

Trieste, Italy: Ivan DonatiUniversity of

Alberta, Canada: Greg

Korbutt

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