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1
Improved alginates for cellencapsulation by the use ofenzymatic engineering
Berit Løkensgard Strand, Ph.D.Neuchatel July 6th, 2005
Neuchatel July 6, 2005
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Microcapsules in cell therapy
(Lanza et al. 1999)
Immune cells
Immunoisolation:
Cells Capsule
Nutrientsand oxygen
Cellproducts
Insulin
Graft rejection +autoimmunedisease
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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
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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
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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.
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In vivo (syngraft):Transplanted ECislets are able toreversehyperglycemia indiabetic animals
In vitro:Encapsulated isletsfunction as non-encapsulatedislets
Lim and Sun, Science 1980
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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
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Important Capsule Properties
• Stability• Permeability• Size• Biocompatibility
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CaCl2
AlginateCells
Formation of Ca-alginate gel beads
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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
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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:
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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
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Important Capsule Properties
• Stability• Permeability• Size• Biocompatibility
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CaCl2
AlginateCells
Formation of Ca-alginate gel beads
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• 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
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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
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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
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Addition of polycation increases capsulestability and reduces capsule permeability
Polycation(Polylysine (PLL,PDL), chitosan, etc.)
Alginate
Ca-alginate gel beads
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Alginate-polycation microcapsules
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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
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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
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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
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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
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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
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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
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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
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-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
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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
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-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
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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
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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
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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
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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)
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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)
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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
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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
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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
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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)
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Rejection of encapsulated graft
Exposedpolycation
Mechanicalfailure
Insufficientimmune protection:• Protruding cells• Permeablemembrane
Alginate
Alginate gel PolycationCells
Leaking materials:- cell products- PLL- high M-alginate- impurities
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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:
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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
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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
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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.)