1
Improved alginates for cell
encapsulation by the use of
enzymatic engineering
Berit Løkensgard Strand, Ph.D.
Neuchatel July 6th, 2005
Neuchatel July 6, 2005
Microcapsules in cell therapy
Immunoisolation:
Nutrients
and oxygen
Immune cells
Graft rejection +
autoimmune
disease
Cell
products
Insulin
Cells
Capsule
(Lanza et al. 1999)
Insulin
Oxygen and
nutrients
Waste products
Complement
components
Fibroblast
c
c
Capsule
membrane
Y
Autoimmune
antibodies
Cell
Cytokines, free radicals
reactive oxygen- and nitrogenintermediates
Y
Y
Y
YY
Y
vv
Y Y
c
c
Secreted proteins
Macrophages
Natural
occurring
antibodies
Antibodies
Antigenpresenting cells
Cytotoxic cells
Lymfokines
T-cells
B-cells
Cellular response
Antibody-producing cells
Humoral response
Encapsulation procedure – the
starting point
• For non-proliferating tissue or cells,
a mild encapsulation procedure is
needed (e.g. Pancreatic islets,
stem cells).
• For proliferating tissue, a mild
encapsulation procedure is not that
important since dead cells can be
replaced by new living cells
History of microencapsulation (EC)
• 1964: First description of
microencapsulation by T.M.S. Chang.
• 1980: Microencapsulated islets as
bioartificial pancreas by F. Lim and A.
M. Sun.
In vitro:
Encapsulated islets
function as nonencapsulated
islets
In vivo (syngraft):
Transplanted EC
islets are able to
reverse
hyperglycemia in
diabetic animals
Lim and Sun, Science 1980
History of microencapsulation
in transplantation
Since then (1980-2005):
Capsule characterisation (and improvements):
- stability
- permeability
- biocompatibility
Technical improvements:
- Bead generators for formation of small and
evenly sized beads
- Purification of materials
Transplantation:
- Allo- and xenotransplantation
- Larger animal models
- Clinical trials
Problem:
- Promising highlights but problems
with reproducing results
Important Capsule Properties
•
•
•
•
Stability
Permeability
Size
Biocompatibility
Formation of Ca-alginate gel beads
Cells
Alginate
CaCl2
Alginate
H
COOH
H
OH
H
O OH
OH
OH
OH
H
H
COO -
G:
H
H
H
H
-L-Guluronic acid (G)
-D-Mannuronic acid (M)
1C
4
O OH
COOOH OH
- OOC
OH
OH O
COO
HO
O
OH
OH
O
O
O
O
COO
HO
M : 4C1
O
-
O
O
OH
O
-
OH
OH
G
COO
-
G
OH
M
M
G
GM M M MGGGGGGGMGMGMGMGM M M M M MG
M - block
G - block
MG - block
M - block
Alginate properties depend on
alginate composition
• Alginates gel forming properties with divalent cations
depend on the G-content as the G-blocks specifically
binds the divalent ions:
¯OOC
Ca2+
OH
OH
O
O
O
OH
G
OH
¯OOC
G
O
Ca2+
Alginate sources
and composition
Alginate source
GM
FGG FMM F
FMG FGGG FGGM FMGM NG>1
FG
FM
Durvillea antarctica
0.32
0.68
0.16
0.51
0.17
0.11
0.05
0.12
4
Macrocystis 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
L. hyperborea, outer cortex
0.71
0.29
0.57
0.16
0.13
0.54
0.03
0.10
20
Algal alginates:
Bacterial alginates:
Pseudomonas sp.
Azotobacter vinelandii
FG
0 - 0,5
0,10-0,85
FGG
0
0,02-0,85
Important Capsule Properties
•
•
•
•
Stability
Permeability
Size
Biocompatibility
Formation of Ca-alginate gel beads
Cells
Alginate
CaCl2
What Determines
Important Capsule Properties
• 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
Stability in saline solution
- Swelling of alginate beads
High-G alginate (69% G)
Diameter (µm)
50mM CaCl2
High-M alginate (43% G)
50mM CaCl2 +
1mM BaCl2
10mM BaCl2
1000
1000
900
900
800
800
700
700
600
600
500
500
400
0
1
2
3
4
5
6
7
50mM SrCl2
400
0
1
2
Change of NaCl-solution
3
4
5
6
7
The alginate distribution in the gel
depends on the gelling conditions
50mM CaCl2
in 0.3M mannitol
50mM SrCl2
in 0.3M mannitol
Intensity profile
10mM BaCl2
in 0.3M mannitol
Intensity profile
Intensity profile
250
250
250
200
200
200
150
150
150
100
100
100
50
50
50
0
0
0
100
200
300
400
Distance (µm)
500
600
0
100
200
300 400
500
Distance (µm)
600
700
0
0
100
200
300
400
500
Distance (µm)
600
700
Addition of polycation increases capsule
stability and reduces capsule permeability
Ca-alginate gel beads
Polycation
(Polylysine (PLL,PDL), chitosan, etc.)
Alginate
Alginate-polycation microcapsules
Reduction in size reduce capsule
stability
Because more of the gel is exposed to the surface of a small gel than a larger
gel, smaller gels are more vulnerable to destabilisation than larger gels:
Destabilization upon PLL-exposure (200µm beads):
Fraction of intact capsules (%)
100
80
Mannitol wash,
Exposure to 0.10% PLL
60
Saline wash,
Exposure to 0.05% PLL
40
Saline wash,
Exposure to 0.10% PLL
20
0
0
4
8
12
16
Time of exposure to PLL (min)
What Determines
Important Capsule Properties
•
Stability
• Permeability
•
•
Size
Biocompatibility
• Alginate gel: 1-2% alginate (98 %
water/buffer) gives high diffusion
rates for small molecules such as
oxygen and glucose
• Composition: High-G alginates
gels are more permeable than
high-M alginate gels
• Adding a polycation layer
reduces the permeability of the
microcapsules
What Determines
Important Capsule Properties
•
•
Stability
Permeability
• Size
•
Biocompatibility
• Reduction in size increase the
diffusion of oxygen and nutrients
to the EC cells
• New technology reduce the size
of high viscous droplets, hence
make beads about 150-200 μm
in diameter with a narrow size
distribution
• Reduction in size decrease the
capsule stability
What Determines
Important Capsule Properties
•
•
•
Stability
Permeability
Size
• Biocompatibility
• Alginate is a non-toxic polymer
• In transplantation: Cells growing
on the capsule surface reduce
the diffusion of nutrients and
oxygen to the EC cells, and may
secrete products that harms the
EC cells
• This fibrotic overgrowth may be
caused by
• products secreted from the EC cells
• the surgical procedure
• endotoxins in the materials
• the polycation
Fibrosis on empty alginate-PLL-alginate capsules
is dependent on the poly-L-lysine (PLL) coating
0.1%PLL exp.10min
Capsules without fibrosis:
0%, n=6
0.05%PLL exp.5min
Capsules without fibrosis:
91 ± 5%, n=3
without PLL
Capsules without fibrosis:
91 ± 6%, n=3
Effects of PLL on tumor necrosis factor (TNF)
production and necrosis in monocytes
TNF
80
60
1000
40
100
Necrosis
20
10
0
10
100
Concentration of PLL (g/ml)
% necrosis
TNF (pg/ml)
10000
Alginate properties depend on
alginate composition
MECHANICAL
PROPERTIES
(gel strength)
DIFFUSION
PROPERTIES
(porosity, charge)
BIOLOGICAL
ACTIVITY
COMPOSITION
+ SEQUENCE
TRANSPARENCY
SWELLING / SHRINKAGE
CHEMICAL
STABILITY
(towards ions and
calcium chelators)
CHARGE DENSITY
binding of polycation
Epimerases catalyze the conversion
of M to G in the alginate chain
HO
O
HOOC
OH
O
HOOC
O
HO
M
HOOC
OH O
HO
O
HOOC
M
OH
O
OH O
O
HO
O
M
-D-ManpA
M
Mannuronan C-5 Epimerase,
(AlgE4)
HOOC
HO
O
HOOC
O
OH
O
OH
O
OH
HOOC
HO
O
HOOC
O
OH
OH
O
O
O
OH
M
G
M
G
-L-Gul pA
Activities
Epimerases
R4
G-blocks, MG-blocks
AlgE1
A1
R1 R2
R3 A2
AlgE2
A1
R1 R2
R3 R4
AlgE3
A1
R1 R2
R3 A2
AlgE4
A1
R1
AlgE5
A1
R1 R2
R3 R4
G-blocks
AlgE6
A1
R1 R2
R3
Long G-blocks
AlgE7
A1
R1 R2
R3
Lyase activity
+ G-blocks, MG-blocks
Short G-blocks
R4 R5 R6 R7
--MG-blocks
A - 385 amino acids
R - 155 amino acids
(Ertesvåg et.al., Glærum et.al.)
HOOC
HO
O
HOOC
OH
O
O
HO
HOOC
OH O
HO
O
O
HOOC
OH O
O
HO
O
OH
-D-ManpA
M
M
M
C-5 Epimerase
M
AlgE4
HOOC
HO
O
HOOC
O
OH
O
OH
O
OH
HOOC
HO
O
HOOC
O
OH
O
OH
O
O
OH
M
G
M
G
-L-GulpA
Capsules of epimerised alginates are smaller
And more inhomogeneous than capsules
from the original alginate sample
B
A
Profile
Profile
Intensity
Int ensity
250
250
200
200
150
150
100
100
50
50
0
0
100
200
300
400
Distance (µm)
500
600
d = 620 μm
V = 0,12 mm2
700
0
0
100
200
300
Distance (µm)
400
500
d = 550 μm
V = 0,09 mm2
600
Stability in saline solution
- Swelling of alginate beads
M.pyrifera
M.pyrifera + AlgE4
5
4
700
V/Vo
Diameter (µm)
800
600
500
3
2
1
400
0
0
1
2
3
4
5
6
7
0
1
2
Change of NaCl-solution
3
4
5
6
7
Stability against osmotic pressure
- Swelling in ion free water
L.hyperborea stipe
L.hyperborea stipe + AlgE4
160
% Increase of capsule diameter
Fraction of intact capsules
1,2
1
0,8
0,6
0,4
0,2
0
0
20
40
60
140
120
100
80
60
40
20
0
0
Time [min] in water
20
40
60
Permeability (IgG, 150 kDa)
Bound IgG [cpm]
Ca/Ba-alginate beads are less permeable to IgG
after epimerisation with AlgE4
Positive control
Unspesific binding
Original alginate
Epimerised alginate
2500
2000
1500
1000
500
0
Controls
M. pyrifera
(43% G)
L. hyperborea
leaf (52% G)
L. hyperborea
stipe (65% G)
Permeability (TNF, 55 kDa)
Bound TNF [cpm]
Alginate-PLL-alginate capsules are less permeable
to TNF after epimerisation
Positive control
Unspesific binding
Original alginate
Epimerised alginate
30000
25000
20000
15000
10000
5000
0
Controls
M. pyrifera
43% G
L. hyperborea
leaf (52% G)
L. hyperborea
stipe (65% G)
Porosity of alginate-polylysine-alginate
Capsules to TNF
% binding (TNF)
100
High-G, PLL
High-G + AlgE4, PLL
80
High-G, PDL
60
High-G + AlgE4, PDL
40
20
0
0
0,05
Conc. of polycation (%)
0,1
Stability against osmotic pressure
Fraction of intact capsules
- Swelling in ion free water
High-G:
High-G + AlgE4:
1,2
1,2
1,0
1,0
0,8
0,8
0,6
0,6
0,4
0,4
0,2
0,2
0,0
0,035%
PDL
0,05%
PDL
0,05%
PLL
0,0
0
20
40
60
0
20
Time [min] in water
40
60
MG coating-alginate binds better to the alginate-PLL
capsule than block alginate and poly-M
µg coating alginate / ml capsules
poly-M:
poly-MG:
FG = 0.06
Block-alginate:
FG = 0.41
FG = 0.45
250
200
150
100
50
0
0
2
8
15
Time of storage (days)
21
Coating the PLL-layer with poly-MG alginate
reduces the overgrowth on implanted empty capsules
70
Glucose oxidation
(pmol/10 capsulesx90min)
350
Retrieval (%)
60
50
40
30
20
10
0
Standard Poly-MG
coating
High-G High-G
(epim.)
300
250
200
150
100
50
0
Standard Poly-MG High-G High-G
coating
(epim.)
Rejection of encapsulated graft
Alginate gel Polycation
Cells
Alginate
Leaking materials:
- cell products
- PLL
- high M-alginate
- impurities
Exposed
polycation
Mechanical
failure
Insufficient
immune protection:
• Protruding cells
• Permeable
membrane
CONCLUSION (1)
Epimerisation of the core alginate with the alternase
AlgE4 gives:
Smaller capsules
Reduced porosity
Increased resistance to swelling
Stronger gel
By epimerising the core alginate we are able to reduce
the toxic PLL layer and still keep the stabile and immune
protective behavior of the capsule
CONCLUSION (2)
Epimerisation of the coating alginate with the alternase
AlgE4 gives:
Better binding to the PLL-layer
Reduced overgrowth on implanted empty capsules
By epimerising the coating alginate we are able to coat
the toxic PLL layer better and thus increase the
biocompatibility of the capsules
THE TRONDHEIM BIOENCAPSULATION
GROUP
NTNU:
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)
www.alginatecapsules.com
International Collaboration
University of Uppsala, Sweden:
Department Cell Biology:
Arne Andersson (Prof)
Stellan Sandler (Prof)
University of Atlanta, Canada:
Department Surgery:
Ray Rajotte (Prof)
Greg Korbutt (Ass. Prof)
Igor Lacik (Prof, Polymer Institute of the Slovak Academy of Sciences)
Dr. David Hunkeler (AQUA+TECH Specialties S.A.)