Role of polymer chemistry in cell encapsulation
by polyelectrolyte complexation in biomedical
and biotechnological applications
Igor Lacík
Polymer Institute of the Slovak Academy of Sciences
Dept. of Special Polymers and Biopolymers
Bratislava, Slovakia
igor.lacik@savba.sk
www.polymer.sav.sk/lacik.html
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
OUTLINE
• ENCAPSULATION
Why polymer chemist(ry) should enter encapsulation?
• POLYMERS IN CAPSULES
Polyelectrolytes as a capsule material
Case 1: Pros and cons of Alginate-based capsules
Case 2: Alternative capsules “PMCG”
Process, mechanism, understanding and optimization
• APPLICATIONS – Biomedicine & Biotechnology
• CONCLUSIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Why polymer chemist(ry) should enter…
Premise of encapsulation:
SEMIPERMEABLE MEMBRANE
made of POLYMERS
• to concentrate encapsulated material
• to protect encapsulated material
• to release encapsulated material in a controlled way
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Why polymer chemist(ry) should enter…
BIOMEDICINE
immunoprotection
BIOTECHNOLOGY
specifically catalyzed reactions
islets of Langerhans
diabetes treatment
immunocytes
glucose, O2,
nutrients
chemistry
food technology
pharmaceuticals
cosmetics
Reactant A
SEMIPERMEABLE
MEMBRANE
therapeutic
products
(insulin)
antibodies
•• still
still in
in development
development stage
stage
•• aa lot
lot of
of involvement
involvement (companies,
(companies, universities)
universities)
igor.lacik@savba.sk
Reactant B
CELLS
(enzymes)
Product
•• already
already as
as industrial
industrial process
process
Neuchâtel, Switzerland,,July 6-7 2005
Detour #1: pancreas (whole organ) transplantation
USA: Number of organ transplantations in 2002 (24 544)
Port et al. Am J Transplant 2004, 4 (Suppl.9): 7-12
0.1% 4.2%
Success rate of pancreas transplantation
1996-2001
8.6%
20.6%
59.2%
0.2%
<1%
3.7%
1.6%
0.6%
100
90
Success rate (%)
Patients with
Type 1 Diabetes
~ 200 000
80
70
60
50
40
30
20
10
0
Kidney +
1
pancreas
Kidney (14 523)
Kidney+pancreas (902)
Heart (2 111)
Pancreas alone (141)
Intestine (44)
Heart+Lung (31)
Pancreas after kidney (376)
Liver (5 060)
Lung (1 041)
Pancreas after
2
kidney
Pancreas
3
1st year - patient
5th year - patient
1st year - pancreas
5th year pancreas
C.N.Street et al, Int J Biochem Cell Biol 2004
Problems in this strategy of diabetes treatment:
Ødonors
Ø major operation
Ø cost
? TRANSPLANTANTION OF VIABLE CELLS?
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Detour #2: Cell therapy in diabetes treatment
Method
Edmonton protocol
Results
Drawbacks
Shapiro et al. N Engl J Med 2000,343,230
• 100 % of patients: physiolo• cell source (2 donors/1 transplant)
• human islets to liver portal
gical
blood
glucose
level
• immune response (allo-, auto-)
vein (11 000/kg)
in 7 patients within 1 year
• ? long-term effect of immuno• islet isolation protocol!!!
•
follow-ups:
Edmonton
&
tens
of
suppressive drugs
• selection of immunosuppreother
groups
USA/Canada/Europe
ssive drugs!!!
http://www.med.ualberta.ca/islet/
follow up: Bertuzzi et al. Diabetologia 2002,45,77
Stem cells and
precursor cells
• embryonic cells
• adult pancreatic cells
• other sources
Street et al, Int. J. Biochem Cell Biol 2004, 36,667
• successful experiments in mice
• a number of open questions
• cell cluster producing insulin
Cell encapsulation
• autoimmune response
• balance between proliferation and
insulin production
• ? unknown effects (tumors…)
deVos et al, Diabetologia 2002, 45, 159
Orive et al, Nature Med 2003, 9, 104
• immunoisolation of donor
cells behind semipermeable
membrane
• immunosuppression-free
igor.lacik@savba.sk
• clinical test in 1994
• “boom”in 90-ties
• allo- and xenotransplanted
cells to different recipients
• progress???
• definite type and material
remains to be answered
Neuchâtel, Switzerland,,July 6-7 2005
Why polymer chemist(ry
…
chemist(ry)) should enter
enter…
…to designed semipermeable membrane
biomedical applications
Biological criteria
• biocompatibility
• resistance to biodegradation
• non-cytotoxicity
(host & transplanted cells)
Physical and chemical
criteria
• mechanical stability
• chemical stability
• adjustment
size/shape
mechanical properties
permeability
membrane thickness
Process criteria
• homogeneity
• capacity
• reproducibility
• availability
biotechnolog
ical aapplications
pplications
biotechnological
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Why polymer chemist(ry
…
chemist(ry)) should enter
enter…
Semipermeable membrane = polymer
Intravascular
semipermeable
membrane
polyacrylates
cells
? major surgery / thrombosis / retrieval
vein
Macrocapsule
cells
semipermeable
membrane
?
•modified cellulose
•poly(acrylonitrile-co-vinylchloride)
hollow fibres
•teflon planar membranes
•polyesters
•macrobeads of polysaccharides
diffusion / islet necrosis / mechanical properties
hydrogels:
• gelling polysaccharides
polyelectrolyte complex
temperature induced sol-gel process
• solvent extraction/ precipitation
Microcapsule
semipermeable
membrane
cells
?
igor.lacik@savba.sk
retrieval
Neuchâtel, Switzerland,,July 6-7 2005
OUTLINE
• ENCAPSULATION
Why polymer chemist(ry) should enter encapsulation?
• POLYMERS IN CAPSULES
Polyelectrolytes as a capsule material
Case 1: Pros and cons of Alginate-based capsules
Case 2: Alternative capsules “PMCG”
Process, mechanism, understanding and optimization
• APPLICATIONS – Biomedicine & Biotechnology
• CONCLUSIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Capsules by polyelectrolyte complexation
A-
M+
A- M +
+
A- M +
PA
+
X- B +
A-.....B+
X- B +
A-.....B+
X- B +
A-.....B+
PC
=
+
M + X-
Polyanion (PA) and poly- cation
(PC) via (predominantly) LONGRANGE COULOMBIC
INTERACTIONS between
oppositely charged polymers will
ALWAYS (!)
form polyelectrolyte complex (PEC)
PEC
Ø Overlapping fields
(i) general macromolecular chemistry
(ii) specificity of polyelectrolyte chemistry
(iii) hydrogels and physically crosslinked networks
(iv) membranes.
? CAPSULE with adjustable properties
è serving the purpose
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolyte comlexation: How the cells are encapsulated
Polymer
Polymer solution
solution
Cells
Cells
DROPLETS
DROPLETS by:
by:
Drop
Drop formation
formation
iioonn
t
t
a
a
t
ggiiaat
a
a
ttllee
n
n
e
e
GG
Inotech
Inotech
Nisco
Nisco
genialab
genialab
…or
…or
In-house
In-house
made
made
100-102 s-1
Mild,
Mild, fast,
fast, controlled
controlled procedure
procedure
at
at physiological
physiological conditions
conditions
è
è POLYELECTROLYTE
POLYELECTROLYTE
COMPLEXATION
COMPLEXATION
Hydrogel
Hydrogel // membrane
membrane formation
formation
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - categorization
• type of charge – PA vs. PC
charge density
charge distributon
• acid-base characteristics
weak vs. strong acids and bases
• molecular weight
• MWD
• chemical distribution
(homopolym. vs. copolymers)
Many possibilities
igor.lacik@savba.sk
• hydrophobic groups
• H-bonds
• gel-formation in the presence
of simple electrolytes
• origin - synthetic vs.
narural vs. modified
• chain architecture – linear vs.
branched
•monomer type - flexible vs
rigid
ALL FACTORS NEED TO
BE CONSIDERED WHEN
STATING SUITABILITY
OF A GIVEN POLYMER
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - selection
Polyanion vs. Polycation
PA – “biocompatible”
– not critically toxic
– internal polymer
INTERNAL
POLYMER
EXTERNAL
POLYMER
igor.lacik@savba.sk
Sodium alginate
Cellulose sulfate
Carboxymethyl cellulose
Hyaluronic acid
Pectates
..............
Polyacrylic acid
PolyAMPS
………..
PC – decomposition of
cell walls
– positive charge has
to be SATURATED
– external polymer
Chitosan
…………
Poly(L-lysine)
Poly(L-ornithine)
................
Poly(methylen-co-guanidine)
Poly(vinylamine)
Poly(ethylenimine)
Poly(DADMAC)
Poly (N-vinylpyrrolidone)
..............
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - selection
Polyanion vs. Polycation
PA – biocompatible
– not critically toxic
– internal polymer
Sodium alginate
Cellulose sulfate
Carboxymethyl cellulose
Hyaluronic acid
Pectates
..............
PC – decomposition of
cell walls
– positive charge has
to be SATURATED
– external polymer
Chitosan
…………
Poly(L-lysine)
Poly(L-ornithine)
INTERNAL
................
POLYMER Polyacrylic acid
Poly(methylen-co-guanidine)
PEC
Poly(vinylamine)
PolyAMPS
PEC – „non-toxic,
biocompatible“ Poly(ethylenimine)
………..
Poly(DADMAC)
– stability! is critical
Poly (N-vinylpyrrolidone)
è multiple layers PA/PC/PA/PC/PA...
..............
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - selection
Charge density
AIM → HYDROGEL (polym. conc. → 0 %)
- physiological environment for cells
- strict selection of PE’s to balance
interactions between PA and PC
WATER POLYMER STRONG INTERACTIONS → COACERVATE
polym. conc. →100 %
- lost control over membrane properties
Additional factors
è polymer concentrations, pH, ionic strength,
reaction time…
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - selection
Molecular weight and MWD
Critical parameter for
→ solution rheology
→ drop and capsule shape and surface topology
→ mutual diffusion of PA and PC zones (pH, ionic strength)
→ osmotic pressure
→ complex stability (biocompatibility and capsule overall performance
RECOMMENDATION – internal polymer é M.h. (rigid chain, gelled
ionotropically by small ions)
100 – 1000 kDa
– external polymer ê M.h. (flexible chain)
5 – 20 kDa
– MWD
– so far not emphasized
Consider in line with
other???
parameters
– concentration, time of reaction, process parameters...
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Polyelectrolytes - selection
Natural polymers
predominantly used as internal polymers
- advantage – ionotropically formed gels
– diversity
– availability and cost
- drawbacks – purity
– standardization!!!
– overcoming these problems ????
Synthetic polymers
- advantage – “simply” prepared, modified,
purified & characterized
- drawbacks – difficult to mimic, by e.g. secondary
structure, the advantageous
properties of natural polymers
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
OUTLINE
• ENCAPSULATION
Why polymer chemist(ry) should enter encapsulation?
• POLYMERS IN CAPSULES
Polyelectrolytes as a capsule material
Case 1: Pros and cons of Alginate-based capsules
Case 2: Alternative capsules “PMCG”
Process, mechanism, understanding and optimization
• APPLICATIONS – Biomedicine & Biotechnology
• CONCLUSIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in microparticle and capsule formation
OH
COO-Na+
O
O
OH
O
OH
O
OH
guluronic acid
(if >60% high-G alginates)
COO- Na+
+ CaCl2 = gel
HO
O
OH
O
HO
mannuronic acid
(if >60% high-M alginates)
EXTREMELY simple
igor.lacik@savba.sk
Ca2+
COO-Na+
COO- Na+
HO
O
O
egg-box model
may cause a problem if the „polymer nature“
of alginate is underestimated
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in microparticle and capsule formation
OH
COO-Na+
O
OH
O
è
è drops
drops of
of alginate
alginate solution
solution into
into CaCl
CaCl22 solution
solution
O
OH
O
OH
guluronic acid
(if >60% high-G alginates)
COO- Na+
+ CaCl2 = gel
HO
O
OH
O
HO
mannuronic acid
(if >60% high-M alginates)
EXTREMELY simple
igor.lacik@savba.sk
Ca2+
COO-Na+
COO- Na+
HO
O
O
egg-box model
may cause a problem if the „polymer nature“
of alginate is underestimated
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate ionotropic gels
Bead inhomogeneity
Na-alginate
Na-alginate into
into CaCl
CaCl22
Gell density across the capsule
? SA concentration in core vs surface
Polymer concentration è
Crucial for bead/capsule properties
SA
SA diffusion
diffusion towards
towards gelling
gelling zone
zone ??
Inhomogeneous
bead
in bead
Ca2+
SL
O
W
high
high Na+
Homogeneous
bead
original
in soltn.
alginate conc.
FA
ST
low Ca2+
low Na+
distance
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
Lim and Sun, Science 210, 908-909,1980
1st
step:
drops of SA (0.6-0.8%) containing islets of
Langerhans (Wistar rats) gelled by Ca2+ ions
3rd step:
strengthening the membrane
0.2 % polyethyleneimine solution
reaction time ~ 2 - 4 min
2+ solution
Ca
1.5%CaCl solution
SA /Ca2+- PLL/PEI coated bead
2
SA/Ca2+ bead
collection time + gelling time ~ ? + ? min
2nd step:
4th step:
Dissolving SA/Ca2+ gel
sodium citrate solution
~ 700 – 1000 µm
membrane formation
0.02% poly-L-lysine solution
reaction time ~ 5 min
SA-PLL/PEI hollow capsule
reaction time ~ 3-5 min
SA/Ca2+-PLL coated bead
igor.lacik@savba.sk
CELL VIABILITY èfour months in vitro
è3 weeks in vivo
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
Modified protocol of Lim and Sun - till now
1st
step:
drops of SA (1-3 %) containing islets of
Langerhans gelled by Ca2+ ions
3rd step:
saturating residual cationic charge by SA layer
0.1 % sodium alginate solution
reaction time ~ 5-10 min
2+ solution
Ca
1.5%CaCl solution
SA/Ca2+-PLL-SA coated bead
2
SA-Ca2+ bead
collection time + gelling time ~ 5 + 5 min
2nd step:
4th step:
Dissolving SA-Ca2+ gel
Sodium citrate solution
membrane formation
~ 0.02% poly-L-lysine solution
reaction time ~ 5-10 min
SA-PLL-SA hollow capsule
reaction time ~ 5 - 30 min
SA/Ca2+-PLL coated bead
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
Patrick Soon-Shiong et al (VivoRx): a dominant influence on encapsulation community 1990-1998
purified alginate with high G content
(mechanical stability & biocompatibility)
ê
SA/PLL/SA hollow capsules
with
15-20 000 islets/kg (allografts)
TX site: peritoneal cavity
ê
Successful dog trials (1992)
ê
Successful clinical trial
(Lancet 1994)
US Patents 1998-1999
Understanding:
(i) PEC stability
(ii) capsule formation
(Trondheim, Norway) Capsule and membrane
mechanical & chemical stability
• alginate rich in G units
• alginate rich in M units for binding to
PLL has to be present (multi-layer)
• no degelling of core by citrate
• stable Ba2+ gelation to Ca2+
• anisotropic distribution of alginate
in core
• absence of PLL positive charge on
surface → macrocapsules
Boom in the field
1. dispersed
in SA
Difficult / impossible to repeat
igor.lacik@savba.sk
~0.7mm
2. Ca2+
y.2005:
Neuchâtel,
Switzerland,,July 6-7 2005
y.2005: clinical
clinical trials
trials www.amcyte.com
www.amcyte.com
Sodium alginate in capsules: history to presence
MODIFICATIONS & “IMPROVEMENTS” TO SA/PLL/SA CAPSULE
→ suppress islet necrosis, improve long-term biocompatibility and durability
SIZE
(diffusion/site of Tx/volume…)
ANIMAL MODELS
ALGINATE TYPE
(chemical composition)
MEMBRANE CHEMISTRY
(biocompatibility)
???
SITE of Tx
(vascularization/explantation/safety…)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
MODIFICATIONS
& “IMPROVEMENTS”
TO SA/PLL/SA
CAPSULE
• high-M
alginate is more suitable
than high-G
yet biocompatibility
→ suppress islet necrosis,
improve
long-term
biocompatibility
durability
is an issue
of purity
(endotoxins,
mitogens,and
phenolic
compounds)
Klöck et al. 1997
• high-M alginate is more suitable than high-G for SA/PLL/SA
SIZE
capsules due
to efficient binding to PLL
(diffusion/site
Tx/volume…)
deVos et al.of(1996-2005)
• high-G alginate should be the main constituent
SA/PLL/SA
ANIMALinMODEL
capsules for their stability
ALGINATE TYPE
Soon-Shiong et al (1998)
(chemical composition) • high-G alginate is more suitable than high-M for SA/PLL/SA
capsules due to improved mechanical stability
Halle et al (1999)
???
MEMBRANE CHEMISTRY
• high-G alginate is more suitable than high-M
for SA-Ca 2+ beads
(biocompatibility) deVos et al. (1996-2005)
• high-M alginate is more suitable than high-G for SA-Ba 2+ beads
Weir et al. (2001-2005)
SITE→ofbiocompatibility
Tx
• high-G vs high-M
is an issue of purity
(vascularization/explantation/safety…)
Orive et al. (2002)
• purity may not be a major issue → for PEC capsules, endotoxins
as polyanions are captured by cationic charge of membrane
Wandrey (1999)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
• replacing PLL by poly-L-ornithine (stable complex with
SA) Calafiore
et al (1995-presence,
clinics) CAPSULE
MODIFICATIONS
& “IMPROVEMENTS”
TO SA/PLL/SA
• replacing
PLL
by aminopropyl
(stable complex,
sol-gel)
→ suppress islet
necrosis,
improve
long-term silicate
biocompatibility
and durability
Sakai et al (2001 - presence)
• SA (macro)bead containing SA-PLL-SA microcapsules
(clinics: www.amcyte.com)
SIZE
• avoiding
PEC
– formation of stable SA/Ba2+
(diffusion/sitemembrane
of Tx/volume…)
beads Weir et al (2001 - presence)
ANIMAL
MODEL
• coating layer based on the PEG chemistry grafting
PEG
on
ALGINATE TYPE PLL Hubbell (1992); presence devoted to PEG chemistry
• etc.
(chemical composition)
MEMBRANE CHEMISTRY
(biocompatibility)
???
SITE of Tx
(vascularization/explantation/safety…)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
MODIFICATIONS & “IMPROVEMENTS” TO SA/PLL/SA CAPSULE
→ suppress islet necrosis, improve long-term biocompatibility and durability
SIZE
(diffusion/site
of Tx/volume…)
• peritoneal
cavity (simple,
fast, high capacity, retrieval?)
• under capsule kidney (localization, retrievalANIMAL
if needed)
MODELS
• portal vein of liver (safety?)
ALGINATE TYPE
• spleen vein (safety?)
(chemical composition)
• subcutaneously (geometry?)
•…
???
MEMBRANE CHEMISTRY
(biocompatibility)
SITE of Tx
(vascularization/explantation/safety…)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
MODIFICATIONS & “IMPROVEMENTS” TO SA/PLL/SA CAPSULE
→ suppress islet necrosis, improve long-term biocompatibility and durability
SIZE
(diffusion/site of Tx/volume…)
ANIMAL MODEL
Calafiore
et
Calafiore
et al
al 1995
1995 –– 2003:
2003:
ALGINATE
TYPE
• medium
medium and
and conventional
conventional size
size microcapsules
microcapsules
(chemical •composition)
600
600 –– 1000
1000 µm
µm
400
400 µm
µm
•• coherent
coherent microcapsules
microcapsules
smaller
smaller is
is better,
better, but
but was
was not
not proven
proven for
for dogs
dogs
Halle
Halle et
et al
al 1996
1996 –– 1999:
1999:
???
MEMBRANE
CHEMISTRY
recommended
<< 350
recommended size
size of
of microcapsules
microcapsules
350 µm
µm
(biocompatibility)
more
more biocompatible
biocompatible than
than conventional
conventional size
size
deVos
deVos et
et al
al 1996
1996 –– 2002:
2002:
large
~~ 800
large diameter
diameter
800 µm
µm
SITE
of
Tx
small
~~ 500
small diameter
diameter
500 µm
µm
(vascularization/explantation/safety…)
larger
larger is
is better
better == aa more
more adequate
adequate islet
islet encapsulation
encapsulation
Strand
Strand et
et al
al 2002:
2002:
larger
~~ 500
larger diameter
diameter
500 µm
µm
small
~~ 200
small diameter
diameter
200 µm
µm
islets
islets in
in vitro
vitro == no
no difference
difference between
between sizes
sizes of
of capsules
capsules at
at
adjusted
adjusted conditions
conditions for
for capsule
capsule formation
formation
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
MODIFICATIONS & “IMPROVEMENTS” TO SA/PLL/SA CAPSULE
→ suppress islet necrosis, improve long-term biocompatibility and durability
SIZE
(diffusion/site of Tx/volume…)
•• typical
–– normal
typical animal
animal donors/hosts
donors/hosts
normal mice
mice
NOD
NOD
ALGINATE TYPE
–– rats
rats
(chemical composition)
–– pigs
pigs
–– dogs
dogs
•• alloallo- vs.
vs. xenotransplantations
xenotransplantations
MEMBRANE CHEMISTRY
(biocompatibility)
•• note
note that
that successful
successful “Edmonton
“Edmonton protocol”
protocol” could
could avoid
avoid
ANIMAL MODEL
???
using
using animal
animal models
models before
before the
the clinical
clinical trials
trials
SITE of Tx
(vascularization/explantation/safety…)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
&
“IMPROVEMENTS”
TO SA/PLL/SA CAPSULE
•• smoothness
vs.
physical
smoothnessMODIFICATIONS
vs. roughness,
roughness, i.e.
i.e.
physical imperfections
imperfections
→ suppress
necrosis, is
improve
→
opinion
smoother
better
→ general
general
opinionislet
smoother
is
betterlong-term biocompatibility and durability
→
→ possibly
possibly “just
“just rough”
rough” is
is better
better (Prof
(Prof Hilborn,
Hilborn, Uppsala)
Uppsala)
è
è ???
??? Quantification
Quantification
SIZE
(diffusion/site of Tx/volume…)
2+ vs
2+
•• Ba
Ba2+
vs Ca
Ca2+
ANIMAL MODEL
→
→ the
the gel/capsule
gel/capsule chemical
chemical stability
stability
2+
ALGINATE TYPE
è
è ???
??? Toxicity
Toxicity of
of Ba
Ba2+
(chemical composition)
•• homogeneous
homogeneous vs.
vs. heterogeneous
heterogeneous capsule
capsule core
core
→
→ meet
meet both
both opinions
opinions
???
è
è ???
??? real
real evidence
evidence
MEMBRANE CHEMISTRY
(biocompatibility)
•• homogeneous
homogeneous vs.
vs. heterogeneous
heterogeneous capsule
capsule core
core
→
→ meet
meet both
both opinions
opinions
SITE of Tx
è
è ???
??? real
real evidence
evidence
(vascularization/explantation/safety…)
•• difference
difference in
in modulus
modulus of
of tissue
tissue and
and material
material
è
è Prof
Prof Hilborn,
Hilborn, Uppsala
Uppsala
•• etc
etc
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
MODIFICATIONS & “IMPROVEMENTS” TO SA/PLL/SA CAPSULE
→ suppress islet necrosis, improve long-term biocompatibility and durability
SIZE
(diffusion/site of Tx/volume…)
ANIMAL MODEL
ALGINATE TYPE
(chemical composition)
MEMBRANE CHEMISTRY
(biocompatibility)
???
SITE of Tx
(vascularization/explantation/safety…)
CIRCLE OUTCOME: A VERY POOR LEARNING CURVE
è struggles for one searching “some degree of exactness”
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Sodium alginate in capsules: history to presence
Enzymatically tailor-made composition of sodium alginate
by group of Prof. S. Bræk, Trondheim Group
mannuronan C-5 epimerase
J Biomed Mater Res 64A, 540-550 (2002)
Effect on
è chain flexibility
è packing density
è interactivity
Improvement
è higher stability
è lower permeability
è core and coat material
→
→ aa new
new hope
hope for
for alginate-based
alginate-based capsules
capsules
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Detour #3: search for exactness (IL’s another field)
IUPAC activities (www.iupac.org/projects/2004/2004-034-1-400.html )
Availability and precision of rate coefficients in free-radical polymerization
ln kp (L·mol-1·s-1)
Arrhenius plot for propagation rate coefficient for methyl methacrylate
9
beginning of 90-ties
benchmark
benchmark values
values
based
based on
on data
data from
from
aa number
number of
of laboratories
laboratories
7
6
5
4
2.6
2.8
3.0
3.2
3.4
3.6
1000 T-1 / K-1
1.
2.
3.
end of 90-ties
8
3.8
2.8
3.0
3.2
3.4
3.6
3.8
4.0
1000 T-1 / K-1
achieved after recognizing drawbacks and critical discussion and setting
consistency criteria
difficult topic but seems a simpler problem than capsule formation
in encapsulation field, critical discussion might be (is) missing,
“ blaming nature” for non-consistency
èprimary
èprimary principle:
principle: consistency
consistency
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Comments on process of capsule formation by PEC
(Lim & Sun, 1980)
1st step:
drops of SA (0.6-0.8%) containing islets of
Langerhans (Wistar rats) gelled by Ca2+ ions
SIMPLE è VASTE AMOUNT OF PAPERS
è ANYBODY CAN DO IT
è ANYBODY CAN REPEAT IT
2+ solution
Ca
1.5%CaCl solution
2
SA/Ca2+ bead
collection time + gelling time ~ ? + ? min
2nd step:
membrane formation
0.02% poly-L-lysine solution
reaction time ~ 3-5 min
SA/Ca2+-PLL coated bead
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Comments on process of capsule formation by PEC
Polyelectrolyte-based beads and capsules
(i) time of droplet collection
(ii) time of gel/membrane formation …
i.e. discontinuous process
take the literature on PEC capsule and find
this (logically required) information
50 randomly selected papers (>y.1999)
30%
6%
No inormation at all
?
igor.lacik@savba.sk
64%
Collection time
Reaction time
• trivial and/or unimportant information?
• confidential information?
• hard to define?
è comparison among the
data/groups is questionable
Neuchâtel, Switzerland,,July 6-7 2005
Comments on process of capsule formation by PEC
collection & reaction times
A wrong observation: beads formed by reaction of Ca2+ with sodium
alginate in a couple of seconds and a couple of minutes look similar
BUT are not identical
•Collection time
•Reaction time
5 minutes
5 minutes
ð Reaction time for individual capsules varies between 5 to 10 minutes
Polymer network
Øgel density
Øeffective molecular weight btw.
crosslinks
Øgel thickness
Øswelling
Ø…..
Capsule
• permeability
• mechanical stability
• chemical stability
• mass transfer
• degree of swelling (size)
• post-reactions (PLL, coat…)
• cell environment
•….
Averaged properties a difficult to judge the capsule/cell
performance (repeatability; discarded systems)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Comments on process of capsule formation by PEC
…limitations in SA-PLL-SA (type of) capsule:
• capsule inter- and intrabatch homogeneity
CaCl2 solution
(GELLING REACTION)
NaCl solution
(WASH & POOL COLLECTED BEADS)
PLL solution
(MEMBRANE FORMATION)
– Ca2+ exchanged by Na +
– time dependent
Capsule wash
ACTIVE STEP
(Strand et al 2003)
Further exploration for capsule type with:
• “independent” adjustment of membrane properties?
(mechanical properties, thickness, permeability, smoothness)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
OUTLINE
• ENCAPSULATION
Why polymer chemist(ry) should enter encapsulation?
• POLYMERS IN CAPSULES
Polyelectrolytes as a capsule material
Case 1: Pros and cons of Alginate-based capsules
Case 2: Alternative capsules “PMCG”
Process, mechanism, understanding and optimization
• APPLICATIONS – Biomedicine & Biotechnology
• CONCLUSIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
“PMCG” microcapsule: basic chemistry
Vanderbilt University, TN
5 components interact in one step
CH2
Polyanion
solution
0.6-1.2 %
Polycation
solution
C
NH
NH
n
Poly(methylene-co-guanidine)
PMCG
Sodium
alginate
(high M)
+
CaCl2
0.6-1.2 %
NH
Sodium
cellulose
sulfate
NaCl
1-2 %
0.6-1.2 %
0.9%
Complex stability in PBS (after Ca2+ removal)
unstable
igor.lacik@savba.sk
stable
highly stable
Neuchâtel, Switzerland,,July 6-7 2005
“PMCG” microcapsule: basic chemistry
Polyanion soltn.
high viscosity (SA)
&
cellulose sulfate (CS)
ê
Polycation soltn.
poly(methylene-co-guanidine)
&
CaCl2 (gelling cation)
&
NaCl (anti-gelling cation)
igor.lacik@savba.sk
• SA/CS ratio - specifically interacting
polyanions with components of PC
solution
• CaCl2 - gelling with SA è spherical
shape
• PMCG - low M.w. (Mn = 4500 g/mol)
and high charge density è high mobility
and reactivity, strongly interactive with CS
• competitive reaction between
PMCG and Ca2+ with the anonic sites
• anti-gelling Na+ cations tune the
gelling reaction
• reaction is fast è a few tens of seconds
• 1-step process è no changes of
membrane introduced between gel bead
and membrane formation
Neuchâtel, Switzerland,,July 6-7 2005
“PMCG” microcapsule: process of preparation
Discontinuous (beaker) collection
typically applied to the PEC-made capsules
• nominal size of droplets generated at
~ 3000/min by air-stripping. size ~ 0.8 mm
• 0.6 % SA + 0.6 % CS in PBS
• 2.2 % PMCG, 0.6 % CaCl2, 1.8 % NaCl in H2O
Collection time:
Reaction time:
30 s
+ 30 s
0.5 mm
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
“PMCG” microcapsule: process of preparation
1-step CONTINUOUS process
Beaker: collect 30’’+ react 30’’
POLYANION
(+ cells)
AIR
air-stripping
nozzle
Reactor 30’’
POLYCATION
drops
capsule formation
Reactor 60’’
multi-loop
reactor
Multiloop reactor
each capsule is exposed to
the same reaction conditions
igor.lacik@savba.sk
final
capsules
capsule uniformity
(geometry, chemistry)
Anilkumar et al Biotechnol. Bioeng. 75, 581 (2001)
Neuchâtel, Switzerland,,July 6-7 2005
PMCG capsule: membrane formation & mechanical resistance
Conc. (wt.%): SA/CS = 0.6/0.6, PMCG 1.8 %, CaCl2 1.0 %, NaCl 0.9%
Rate of membrane formation
~ tens of second
Mechanical stability
0.06
40
30
0.04
rupture load (g)
membrane thickness (mm)
0.05
0.03
20
0.02
10
0.01
Alginate / Poly-L-lysine capsule
0.00
0
10
20
30
40
0
0.00
0.01
0.02
0.03
0.04
membrane thickness (mm)
reaction time (s)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Permeability of PMCG capsule è inverse SEC
1. Capsules used as a column packing
2. Pullulan standards of different molecular weight injected on column
è molecular weight cut-off (MWCO)
è pore size distribution
~ 20 ml
0.9 % NaCl
0.2 ml/min
1-3 mg/ml
100 µL
MWCO
1
0.8
1-KSEC
column bed
eluent
flow rate
pullulan conc.
inj. volume
0.6
0.4
KSEC = (Vi-V0)/(Vt-V0)
0.2
d(1-KSEC)/d(logM)
Brissova et al Analytical Biochem. 242, 104-111, 1996
0
2
3
4
5
6
log M
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Permeability of PMCG capsule è effect of MW of CS
PA: 0.9% SA, 0.9% CS in 0.9% NaCl
PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7
40’’ reaction time, citrate step
CS in polyanion composition
CS as a coating layer (0.1%, 10 min)
1
1-K SEC
0.8
0.6
760 kDa
36 kDa
360 kDa
41 kDa
190 kDa
1
MWCO
MWCO
CS
CS
-
0.8
1-K SEC
CS
CS
58 kDa
0.4
0.2
0.6
MWCO
MWCO
36 kDa
760 kDa
18 kDa
190 kDa
6 kDa
0.4
0.2
0
0
2.5
3
3.5
4
4.5
log M
5
5.5
6
2.5
3
3.5
4
4.5
log M
5
5.5
6
ð effect of the molecular weight of CS is important
•
network density
•
coating of primary membrane
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Permeability of PMCG capsule è internal polymers
SA in polyanion composition
Polyanion concentration
PA: 0.9% SA, 0.9% CS in 0.9% NaCl
PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7
40’’ reaction time, citrate step
PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7
40’’ reaction time, citrate step
1.0
1
PA
PA
0.6
SA
SA
MWCO
MWCO
430 kDa
36 kDa
300 kDa
60 kDa
0.8
0.6
1-K SEC
1-K SEC
0.8
MWCO
MWCO
0.90/0.90 36 kDa
0.75/0.75 50 kDa
0.60/0.60 80 kDa
0.4
0.4
0.2
0.2
0
0.0
2.5
3
3.5
4
4.5
5
5.5
6
log M
ð refer to Wandrey 2003 – more effect
of SA chemical composition than MW
2.5
3.0
3.5
4.0
4.5
log M
5.0
5.5
6.0
ð effect on MWCO and pore size
distribution
ð correlation to years at Vanderbilt?
! MW (and MWD) affects also other properties than MWCO (stability, rheology…)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Permeability of PMCG capsule è secondary coating
Polyanion Concentration and Secondary Membrane Effects
1.8 wt% PMCG, 1.0 wt% CaCl2, 0.9 wt% NaCl, 1min
Rη
[nm]
0.6 / 0.6
0.9 / 0.9
0.9 / 0.9
0.9 / 0.9
0.9 / 0.9
0.9 / 0.9
230
120
44
19
13.8
3.2
11.7
8.5
5.1
3.4
2.9
1.4
PROTEINS
(globular
conformation)
[kDa]
1 770
750
200
67
44
6.5
molecular weight cut-off
igor.lacik@savba.sk
PLL
PLL 0.01
0.01 wt.%;
wt.%;
33 and
and 4.5min
4.5min
1-KSEC
DEXTRANS
(statistical coil)
[kDa]
1-KSEC
SA / CS
in PA solution
(wt.% / wt.%)
log Rη (Å)
log Rη (Å)
PVAm
PVAm
0.001
0.001 wt.%;
wt.%; 33 min
min
0.1
0.1 wt.%;
wt.%; 55 min
min
Neuchâtel, Switzerland,,July 6-7 2005
Mechanism of capsule formation – a kinetics view
Lacik et al J Microencapsulation 2001
I)
shape relaxation after impact by viscous forces
time ~ 10-3 s
a surface smoothness, sphericity
and permeability
BEFORE IMPACT
AIR
POLYCATION
EFFECT OF GELLING CONDITIONS
II)
AFTER IMPACT
IDEAL RECOVERY
shape relaxation by osmotic swelling
time ~ 10-3 - 100 s
EFFECT OF MW, CONCENTRATION, REACTIVITY
III)
membrane formation
time ~ 100 - 102 s
a required thickness, mechanical and
chemical stability and permeability
OVERALL PROCESS AND CHEMISTRY CONDITIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Shape relaxation by viscous forces
COMMON UNDERSTANDING: NaCl provides physiological osmotic pressure
ADDITIONAL ROLE:
Screening of ionic interactions a effect on
kinetics!
Drop penetration via
air-liquid interface
Before impact
Air
Polycation
solution
After impact
Ideal shape
PE
complexation
Time to reach the “ideal shape” is given by viscous forces
R2
t≈
5ν
 ~ 0.052 ( cm 2 ) 


2 −1
~
1
(
cm
s
)


≈ 10 −3 s
ðEncapsulation process – minimize time needed for shape relaxation:
êdrop size, é viscosity ð OK, but limited by process
slow-down the rate of gel formation !
Kendal et al. 1989
igor.lacik@savba.sk
Antigelling effect of
NaCl
Neuchâtel, Switzerland,,July 6-7 2005
Shape relaxation è gelling conditions [Na+]/[Ca2+]
Optical microscope
a) Immediately after membrane formation
b) After dissolving of internal gel by citrate
a)
a)
b)
b)
0.5 mm
without NaCl in cation solution
0.5 mm
0.9 wt.% NaCl in cation solution
Positive effect on capsule quality
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Shape relaxation è gelling conditions [Na+]/[Ca2+]
Quantification: AFM
10 x 10 µm
LINE PROFILES
0.9 wt% of NaCl
1.0 wt.% of CaCl2
1.0 wt.% of PMCG
4.5 wt% of NaCl
1.0 wt.% of CaCl2
1.0 wt.% of PMCG
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Shape relaxation è gelling conditions [Na+]/[Ca2+]
Effect on permeability
SA/CS = 0.6/0.6 (wt% / wt%), poly (methylene-co-guanidine) 1.0 wt%
CaCl2 (%)
0.60
0.86
1.00
NaCl (%)
0.9
0.9
0.9
[Ca2+ ]/[Na+ ]
exclusion limit [kDa]
0.35
0.49
0.58
97
137
215
ì[Ca2+ ]/[Na+ ] ì MWCO
ì[Ca2+ ]/[PMCG] ì MWCO
Indication
Indication of
of the
the competition
competition processes
processes in
in the
the membrane
membrane formation
formation
Increased
Increased NaCl
NaCl concentration
concentration lowers
lowers the
the PA
PA concentration
concentration at
at the
the drop
drop surface
surface
ð
ð homogeneous
homogeneous vs.
vs. heterogeneous
heterogeneous distribution
distribution of
of polyanions
polyanions
Extremely
Extremely simple
simple adjustment
adjustment of
of MWCO
MWCO
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Shape relaxation è gelling conditions [Na+]/[Ca2+]
Lacik et al J Microencapsulation 2001
Increased ratio Na+/Ca2+ leads to
smoother surface,
but less stable complex
Solution: capsules
are formed continuously in two steps
c2,i
c (1 + k ) − c1,i
= i
k
PC1
high [Na+]
Mixing
Capsules PC1 & PC2
& PC1
PC2
low [Na+]
k is the flow rate ratio
V2/V1 of PC2 and PC1
aCapsules of different
surface quality
igor.lacik@savba.sk
smooth surface
< 5s
Capsules & PC of composition
for membrane at physiological NaCl
stable membrane
tens of seconds
Neuchâtel, Switzerland,,July 6-7 2005
Mechanism of capsule formation – a chemistry view
interactions – dilute solution
composition – real conditions
capsules are made by interactions between:
PMCG + SA
PMCG + (SA-Ca2+)
PMCG + CS
PMCG + SA/CS
PMCG + (SA/CS-Ca2+)
Questions:
• what do we know about interactions?
• can the information on interactions help?
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Interactions in dilute solution
viscometry (Ubelohde viscometer) at 25ºC
+
+
HO -[ CH2 – NH – C – NH –]n- H
relative viscosity
+NH
Mmonmol = 107 g/mol
3.0 charge / unit ?
CH2-OSO3
O
O
HO
O3SO
Mmonmol = 341 g/mol
1.7 charge / unit
COO
equilibrium (electroneutral)
composition
0
1
addition of PE 1 to PE 2
O
O
HO
HO
Mmonmol = 198 g/mol
1.0 charge / unit
expected???
è
è
è
igor.lacik@savba.sk
molecular weight
concentration
ionic strength
PMCG : CS ~ 1 : 2 (xPMCG ~ 0.33)
PMCG : SA ~ 1 : 3 (xPMCG ~ 0.25)
Neuchâtel, Switzerland,,July 6-7 2005
Interactions in dilute solution: PMCG - CS
IONIC STRENGTH
(both polymers 0.01wt.%)
water
0.001M NaCl
0.1M NaCl
Relative viscosity
1.4
1.3
PMCG
e
cha
e
v
i
t
c
ffe
2+
3+
1.2
rg e
1+
PMCG : CS
4:1
1.1
1.0
0
20
40
60
80
100
PMCG (mol %)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Interactions in dilute solution: PMCG - SA
POLYM. CONCENTRATION
(in 0.01 M NaCl)
IONIC STRENGTH
(both polymers 0.008 wt.%)
Relative viscosity
1.7
1.6
1.5
1.4
1+
3+
PMCG : SA
4 :1
1.3
1.2
1.20
Relative viscosity
water
0.001M NaCl
0.01M NaCl
0.1M NaCl
1.8
0.010 wt.%
0.008 wt.%
0.005 wt.%
1.15
1.10
PMCG : SA
4 :1
1.05
1.1
1.00
1.0
0
20
40
60
PMCG (mol %)
igor.lacik@savba.sk
80
100
0
20
40
60
80
100
PMCG (mol %)
Neuchâtel, Switzerland,,July 6-7 2005
Outcomes from studies of interactions in dilute solution
• PMCG is less (electrostaticaly) interactive
than assumed (PMCG effective charge?)
• PMCG content in complex is higher than
stechiometrical.
• Other than (only) ionic interactions are responsible
for complex formation?
• similar PMCG interactivity to both SA and CS
SA + Ca2+
CS + PMCG
SA + PMCG
è Question: complex composition under
REAL conditions?
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Membrane formation under real conditions
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Confocal laser scanning microscopy in membrane characterization
Multi-spectral confocal microscope Zeiss LSM 510-META+Axiovert 200 Mot
Two fluorescence/reflection channels
One transmission channel
32-channel polychromatic detector
488 nm laser line and emission bands 435-485 nm (reflection) and 535-590 (fluorescence)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Confocal laser scanning microscopy in membrane characterization
Fluorescence labels è non-covalent labeling probes after capsule formation (10-7 mol/L)
[
]
HO - CH2 – NH – C – NH – n- H
NH
selected cation probes
(visualisation of negatively-charged polymers)
selected anion probes
(visualisation of positively-charged polymer)
Sensitive
Sensitive to
to (free,
(free, unbound)
unbound) charged
charged polymer
polymer groups
groups
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
CLSM characterization of microcapsule wall – anionic probes
PA: 0.9% SA, 0.9% CS in 0.9% NaCl, PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7 , 40’’ reaction time, citrate step
Reflection è “inner” membrane
A) transmission, B) reflection, C) fluorescence, D) cross-sections (across white line in B,C)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
CLSM characterization of microcapsule wall – cationic probes
PA: 0.9% SA, 0.9% CS in 0.9% NaCl, PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7 , 40’’ reaction time, citrate step
Reflection & fluorescence for non-bulky probes
è “inner” membrane (of anionic nature)
A) transmission, B) reflection, C) fluorescence, D) cross-sections (across white line in B,C)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Dilemma between 1- and 2-step processes
1 – step ð capsular membrane SA/CS – PMCG is made in one step
(by using loop-reactor)
ð intra- and inter-batch homogeneity (geometry and chemistry)
ð 1st step:
2nd step:
2 – step
SA/CS – Ca2+ beads (step consisted of collection + reaction)
exposure to PMCG
ð easy control of membrane thickness and shape
ð may arise a problem with intra- and inter-batch homogeneity
(an issue for large batches!) due to:
Ø differences in collection and reaction times
Ø diffusion of CS between 1st and 2nd steps (CS is not bound by Ca2+)
Ø duration of washing steps
…what can CLSM see?
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
CLSM characterization of microcapsule wall: non-covalent labeling
2-step
2-step process
process
1-step
1-step process
process
PA: 0.9% SA, 0.9% CS in 0.9% NaCl,
PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7
40’’ reaction time
Rhodamine 123
Eosin Y
PA: 0.9% SA, 0.9% CS in 0.9% NaCl,
Step 1: 1.0% CaCl2 0.9% NaCl, pH 7, 40’’
reaction time,
Step 2: 1.2% PMCG, 0.9% NaCl, pH 7, 40’’
reaction time
Rhodamine 123
Eosin Y
Intensity
Intensity
label
Intensity
reflection
reflection
label
label
Intensity
label
reflection
reflection
Distance
Distance
Distance
Distance
è CLSM identifies membrane composition and suggests
mechanism of capsule formation
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Permeability by inverse SEC è 1- vs. 2-step process
PA: 0.9% SA, 0.9% CS in 0.9% NaCl
1-step capsule: 1.2% PMCG, 2.0% CaCl2 0.9% NaCl, pH 7, 40’’ reaction time, citrate step
2-step capsule: 1st step:
2nd step:
citrate
2.0% CaCl2, 0.9% NaCl
1.2% PMCG, 0.9% NaCl
40’’
40’’
1.0
1 -K S E C
0.8 process MWCO
0.6
1-step
52 kDa
2-step
29 kDa
0.4
0.2
0.0
2
2.5
3
3.5
4
4.5
5
5.5
6
log M
MWCO shows a denser network in 2-step process
? a higher CS concentration towards surface ?
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
CLSM characterization of microcapsule wall – covalent labeling
PA: 0.9% SA, 0.9% CS in 0.9% NaCl, PC: 1.2% PMCG, 1.0% CaCl2 0.9% NaCl, pH 7 , 40’’ reaction time
PMCG
PMCG –– rhodamineB-isothiocyanate
rhodamineB-isothiocyanate
igor.lacik@savba.sk
SA
SA –– fluorescein
fluorescein amine
amine
Neuchâtel, Switzerland,,July 6-7 2005
CLSM characterization of microcapsule wall
• extremely useful tool for membrane and capsule
characterization
• understanding mechanism and, hence, control of capsule
formation
• specifically bound labels provide sufficient primary
information; covalently bound labels provide a rigorous
information
• cooperation among devoted people is needed
• future: also for viability studies of encapsulated cells
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
OUTLINE
• ENCAPSULATION
Why polymer chemist(ry) should enter encapsulation?
• POLYMERS IN CAPSULES
Polyelectrolytes as a capsule material
Case 1: Pros and cons of Alginate-based capsules
Case 2: Alternative capsules “PMCG”
Process, mechanism, understanding and optimization
• APPLICATIONS – Biomedicine & Biotechnology
• CONCLUSIONS
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
PMCG capsule for encapsulation of islets of Langerhans
Released insulin (pM/100 cell/min)
Capsules with rat islets
900
freshly isolated islets
freshly isolated islets after encapsulation
encapsulated islets explanted after 9 months
800
700
stimulation
stimulation
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Perifusion time (min)
Capsules
Capsules before
before Tx
Tx to
to C57
C57
mice
mice (A)
(A) and
and explanted
explanted after
after
~~ 99 months
months (B)
(B)
Perifusion in glucose
medium
capsules seem to be biocompatible and cells remain viable
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
PMCG capsule for encapsulation of islets of Langerhans
~ 1000 islets of Langerhans / mouse model intraperitoneally
capsule: 800 µm, membrane ~ 70 µm, MWCO towards dextrans
Neonatal pig islets
NOD mice
MWCO 100 kDa
500
400
300
200
100
(16)
(10)
(11)
(15)
(8)
0
-20
0
20
40
60
80
100
Time after transplantation (days)
120
Glucose concentration (mg/dL)
Glucose concentration (mg/dL)
Sprague-Dawley rat islets
C57 mice
200 mg STZ/kg
MWCO 100-230 kDa
500
400
300
200
100
(9) (7) (6)
(5)
(2)
0
-20
0
20
40
60
80
100
Time after transplantation (days)
The same for NOD mice with rat islets
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
PMCG capsule used in biotechnology
Immobilization of enzymes and whole cells as catalysts for biotransformation
Nocardia tartaricans whole cells with cis-epoxysuccinate hydrolase acitvity
O
O
ONa
ONa
H2O
cESH
COONa
H
HO
OH
H
COONa
O
cis-epoxysuccinate
L-(+)-tartrate
Bucko
Bucko et
et al
al EMT,
EMT, 2005,
2005, 36/1,
36/1, 118,
118, 2005
2005
SA-CS/PMCG capsule vs. typical calcium pectate beads)
Operational stability
140
250
6
200
120
activity 150
(U/mg) 100
time (h) 3
50
2
relative activity (%)
5
4
encapsulated cells
100
80
60
40
1
0
SA-CS/PMCG
CPG
free cells
20
0
SA-CS/PMCG
Storage stability after 35 days
CPG
Time for complete bioconversion
0
0
1
2
3
4
5
6
7
8
9
10
11
cycle number
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Conclusion remarks to polymers used in encapsulation of
biological substances
BIOTECHNOLOGY
• industrially utilized process using mainly
“simple” encapsulation systems (alginates, pectates, PVA)
• ? effect of tight-controlled parameters on efficiency
vs. cost
• a “simple” problem compared to biomedicine
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Conclusion remarks to polymers used in encapsulation of
biological substances
BIOMEDICINE
long-term biocompatibility – capsules tested and tailored
in vitro, the in vivo performance = black-box
• inter-laboratory repeatability is poor
• polymers – lacking characteristics, purity, and
standardization (and fate in vivo after losing performance)
• locus of transplantation?
• final composition of polymeric material?
(microsphere = gelled SA?; microcapsule = ???)
• “academic” approaches – change them to the applicationdriven activities (+ 5 years time…?)
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Concluding message è whether success or failure, always
(for understanding the topic of immobilization of biological substances)
1. THINK OF POLYMERS AND PROCESS, and
2. SEARCH FOR MESSAGES FROM POLYMER CHEMISTRY
©2004,
©2004, www.cavalier.sk
www.cavalier.sk
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Acknowledgement
Polymer Institute SAS, Bratislava (polymers)
G. Kolláriková, M. Danko, E. Hipka, S. Hanzelova, M.Stach, A.
Durisova
International laser center, Bratislava (AFM, CLSM, SIMS)
D.Chorvát, Jr., J. Podskočová
Institute of Chemistry SAS, Bratislava (enzyme engineering)
P. Gemeiner, M. Bučko, A. Vikartovská, J. Nahalka
Institute of experimental endocrinology, Bratislava (islets)
V. Štrbák, J. Benický, M. Najvirtová, Z. Bačová
Slovak grant agency for support of science and technology
APVT, BASF Ludwigshafen, Slovak Grant Agency VEGA, COST 840
igor.lacik@savba.sk
Neuchâtel, Switzerland,,July 6-7 2005
Acknowledgement
Nashville Cell Encapsulation Team, Vanderbilt Uni
(NASA, NIH, NSF)
T. G. Wang
A. V. Anilkumar
M. Brissova
D. Hunkeler
A. Prokop
A. Powers
K. Xu
igor.lacik@savba.sk
PI
reactor design
capsule design, permeability
capsule design
capsule design
medical center
AFM
Neuchâtel, Switzerland,,July 6-7 2005