Functionalization of alginate gel

beads for release control of

macromolecular drugs
Akihiko KIKUCHI
Dept. Mater. Sci. & Technol.
Tokyo University of Science, Japan

Todays topic
1. Background of Alginate
2. Alginate in Medicine
3. Objectives
4. Release Behavior of Model Drugs
5. Modification of Alginate Gels

Background
Alginate: obtained from brown seaweed, algae.
consists of -D-mannuronate and -L-guluronate.
used as food additive (thickener) SAFE MATERIAL
polyguluronate segments form so called egg-box
junctions with divalent cations like Ca2+, Ba2+, Sr2+.
COO-

O
HO

HO
O

O
HO
COOO

HO

COO-

O
HO

HO

O
HO

COO-

HO
O

Poly(D-Mannuronate)
COO-

COO-

HO
O

O
H
O
O
HO

HO

O
O
H
O

O
O
H

O
O
O

COO- HO

Poly(L-Guluronate)

H
O
COO-

Egg-box junction formation of

polyguluronate segments
HO
HO

O
HO

C O

O Ca2+

O
C

HO

C O

O
C

O
OH

O
HO

HO

O
C O

HO

Ca2+

2+
O Ca

O
HO

O
OH

HO

O
OH

Egg-box model for Ca2+ induced dimerization of poly(L-guluronate).

Oxygen atoms (filled circle) involved in chelation.
Ca2+
2+
Ca

2+
Ca

Ca2+

Ca2+

Ca2+

Guluronate with more than 6

units forms egg-box
junctions with calcium ions.

2+
Ca
2+
Ca
Ca2+

Alginate gels in Medicine

Wound dressing because of biocompatibility of
alginate.
Cell encapsulation matrixes: Ca2+-alginate was
used to encapsulate Langerhans islets of
pancreas and coated with poly(lysine) to form
polyelectrolyte complexes and used for treatment
of diabetes patients from early 80s.
Drug delivery matrixes for low molecular
weight drug molecules.

Needs for macromolecular drug carriers

Some biomolecules and biomacromolecules are

released in a certain biological rhythm; e.g. circadian

rhythm, or circalunar rhythm.
insulin, melatonin, estradiol, IL-1, IL-6, -IFN, TNF-,

To achieve drug release mimicking such biological

rhythms, pulsatile drug release system is one of the

possible approaches.

Objectives
To investigate release profile of model macromolecular
drugs; FITC-labeled dextran, from Ca2+-alginate gel beads.
Modification of onset release time through several factors
including, gel bead size, polymer coating on the gel beads.

Conceptual design for onset release control of

macromolecular drugs using Ca2+-alginate gel beads.

Preparation of Ca2+-alginate gel beads

Syringe pump
1.0mL/min
Syringe w/ 2wt%
Na-alginate soln
containing model
drugs

2 wt-% Na+-alginate (MW1.0x105)

aqueous soln w/ FITC-labeled dextran
was dropped into 0.1M CaCl2 soln and
stirred for 24h.
MW of FITC-dextran: 9.4x103~1.45x105
Gel bead size was controlled either by
inner diameter of the needle or flow
rate. To reduce gel diameter, nitrogen
gas flow was also applied.

0.1M CaCl2 soln

Estimation of elution profile for

dextran from Ca2+-alginate gel beads
Release experiments were performed under the protocol
of the 15th Japanese Pharmacopeia.
continuous rotation
at 100rpm

Ca2+-alginate gel beads

0.5g
unmodified
polymer coating
500mL Dulbeccos phosphate buffered
saline soln (PBS-) at pH7.4 and 37C.

thermostated water bath at 37C

Sampling 5mL of PBS at predetermined time

period and measured fluorescent intensity of
FITC-dex with Ex: 495nm, Em: 520nm.

Ca2+-alginate gel disintegration

Ca2+ ion release showed twophase release behavior. In the
first phase (~60%) Ca2+ release
was governed by diffusion
mechanism. Next Ca2+ release
was coincides with alginate
disintegration.
Considering the M/G ratio (=1.3)
of alginate, initially released Ca2+
was from mannuronate units
followed by Ca2+ release from
guluronate units in which Ca2+
ions were interacted through
coordination bonds.

bead diameter:
2.40.2mm
Kikuchi, J. Control. Rel. 47, 21 (1997)

Dextran release from

Ca2+-alginate gel beads
Release behavior of dextran w/
different molecular weight was
examined.
Initial release of low MW dextran
was governed by diffusion, but with
increasing MW, initial release was
suppressed.
High MW dextran release was
synchronized with alginate release.

High MW dextran release was

governed by the disintegration of
Ca2+-alginate gels.

Release of Brilliant Blue

Low MW model drug, brilliant
blue, eluted out of Ca2+alginate gel beads through
diffusion regardless of
alginate MW.

Alginate MW
Circle: 6x104
Triangle: 1x105
Square: 1.4x105

This is in good agreement

with the previous reports for
low MW drugs, like antiinflammatory agent,
dichlofenac.
bead diameter: 2.5mm
Kikuchi, J. Control. Rel. 58, 21 (1999)

Dextran release mechanism

Dextran release was plotted against
the square root of time.
Dextran release did not follow
diffusion mechanism.
In addition, dextran release
occurred in connection to the
alginate release.

bead diameter:
2.40.2mm
Kikuchi, J. Control. Rel. 47, 21 (1997)

Effect of alginate concentration

To control onset release time,
alginate concentration in gel
bead preparation was altered
and release of dextran was
monitored.
Onset release time was
increased for up to 200min with
4% solution with increasing
alginate concentration without
enhancing initial release of
dextran.

High conc. of alginate solution is

viscous and hard to handle. Thus
other method should be considered.

Effect of bead diameter

Beads diameter was changed by needle thickness and flow rate.
dextran release

dextran release from mixture

Time (h)

With bead diameter, onset release time was

extended. Thus by mixing gel beads with
different diameter, dextran release shows three
pulses within 60-240 minutes.

Kikuchi, J. Control. Rel. 47, 21 (1997)

Some problems of Ca2+-alginate gel

beads as drug carrier
Administration route
Oral: Possible for low MW drugs (release pattern: diffusion)
Hard for high MW drugs due to limited uptake from GI tract
and degradation of such drug molecules
Injection: Possible for smaller bead size (faster release pattern)
Hard to extend onset release time due to smaller bead size

New approach to extend onset release time with smaller sizes

is necessary.

Solution for such problems

Need to extend onset release time with smaller bead size
Coating with hydrophilic carboxyl-containing polymers
Bare alginate gel beads

modified alginate gel beads

barrier or
buffering layer
for Ca2+ release

Ca2+-alginate
gel beads

* smaller bead size

* smaller bead size

* faster gel disintegration

* slower gel disintegration

* rapid drug release

* retarded onset drug release

Preparation of carboxyl-containing
water soluble polymers
preparation of 3-carboxy-n-propylacryamide (CNPAAm)
Cl

H2N

H3C

CO

NH

CH3

Ca(OH)2
OH

ice bath

OH

-aminobutylic acid

acryloyl chloride

CO

H
N

CNPAAm

Preparation of poly(N,N-dimethylacrylamide
(DMAAm)-co-CNPAAm) (PDMCP) via free radical
copolymerization using AIBN as radical polymerization
initiator in DMF at 60C for 24h.

CO

NH

n
(CH2)3COOH

P(DMAAm-co-CNPAAm)
(PDMCP)

COOH

n
COOH
Poly(-glutamate) (PGA)

Poly(acrylic acid)
(PAAc)
commercial product

Cumulative release of dextran from unmodified

and modified Ca2+-alginate gel beads
coating polymer; PDMCP: 9.9mol%
cumulative release of FITC-dex
(MW: 1.45x105) (meanSD; n=3)

release rate of FITC-dex (MW:1.45,x105)

100

release (%)

90
80

unmodified

(onset: 80min)

70
60
50

2.5%

(onset: 75min)

40
30

5.0%

20

(onset: 135min)

10
0
0

50

100 150 200 250 300 350 400

time (min)

release rate
release
rate(g/min)
(g/min)

5.0

unmodified

4.5
4.0
3.5
3.0

2.5%
5.0%

2.5
2.0
1.5
1.0
0.5
0.0
0

50

100

150

200 250

time (min)

Polymer coating suppress initial drug leakage.

Onset release time can be extended with polymer coating.
Coating increases release periods.

300

350

400

Preparation of smaller Ca2+-alginate gels

conventional method

proposed method

2.0mL/min

1.0mL/min

N2 flow
of 0.5MPa

2.0 mm

sheath
0.5-1.0 mm

N2 flow

Syringe was connected with thin tubing with needle, which surrounded
with sheath. N2 gas flow was applied through the sheath and alginate
droplet was forced to release from the needle tip. Thus, bead
diameter was regulated from 0.50.1 mm to 2.00.1 mm.

Effect of coating thickness on dextran release

1mm-beads; coating polymer; PDMCP: 23mol%
100

25m-thick coat

75m-thick coat

125m-thick coat

80
9.4x103
60

7.1x104

40
1.45x105
20
0
0

12

16
Time (h)

Effect of coating thickness was examined. Thicker coating

induced retarded onset release time for up to ~50h.
By mixing these gels, appropriate release control should be
achieved modified Ca2+-alginate gel beads.
Iskakov, J. Control. Rel., 80, 57 (2002)

Change in gel bead surfaces during

dextran release
appearance of the gels
before release test

during gel disintegration

Fragmentation of the coating
layer and these remained after
dextran release.

fluorescent image of eroding modified gel beads

fluorescent microscope BX-51

OLYMPUS
objective lens: x10
camera DP-70OLYMPUS)
exposure1/120 sec.

Mechanism of drug release

Two possible mechanism for modified gel
bead disintegration through ionic exchange.
ion exchange

drug release w/ gel

disintegration

1.

2.

dissolution of coating layer

ion exchange

fissure formation on coating

layer
drug release w/ gel
disintegration

gel swelling

Coating layer became fragments during release

experiments and such debris was remained after the
experiment.

Elution behavior of dextran from

PAAc-coated Ca2+-alginate gel beads

PAAc coating effected to retard release

onset time of dextran with increasing
coating polymer concentration.
However, its extension was smaller
than PDMCP.
There is no polymer fragments after
release experiments, suggesting that
all the coated layers were dissolved
during release experiments.

release of FITC-dex (MW: 75,000)

cumulative release (%)

Water soluble poly(acrylic acid) (PAAc)

was coated on the Ca2+-alginate gel
beads and dextran release was
examined.

100
90
80

unmodified

70
60
50
40
30
20
10
0

(onset: 35min)

2.5% PAAc
(onset: 60min)

5.0% PAAc

(onset: 80min)

25

50

75 100 125 150 175 200

time (min)
Coating polymer layers show buffering
effect for Ca2+ ion release.

meanSD (n=3)
bead diameter: 2.00.1mm

Elution of dextran from PGAcoated Ca2+-alginate gel beads

poly(-glutamate) (PGA) is
recently used as degradable
tissue engineering matrix.

PGA coating induced

retarded onset release time
with suppressed initial elution
of dextran (MW7.1x104).
Fragmentation of coating
layer occurred.

100
release (%)

PGA was used as coating

materials.

PGA coating
MW: 1.5106

80
60
40

unmodified
3.0w/v%
5.0w/v%

20
0
0

50

100
tim e (m in)

150

200

Conclusions
Ca2+-alginate gel beads were examined as
possible macromolecular drug carrier.
Alginate with M/G~1.3 showed burst release after
a certain onset release time.
Onset release time can be controlled by gel size,
and polymer coating.
Need to investigate protein release behavior from
these alginate gel beads.

Acknowledgements
y Institute of Advanced Biomedical Engineering and Science,
Tokyo Womens Medical University, Japan
Professor Teruo Okano, Ms. Minako Kawabuchi
Dr. Rinat Iskakov

y Department of Pharmaceutics and Pharmaceutical

Chemistry, Tokyo University of Science, Japan
Professors Kimiko Makino, Hiroshi Terada, Mr. Hirokazu Suzuki

y Department of Materials Science and Technology, Tokyo

University of Science, Japan
Mr. Shohei Ichikawa

Financial supports from the Center for Drug Delivery

Research under the High-Tech Research Center Program, the
Ministry of Education, Sports, Science and Technology (MEXT),
Japan. (Profs. H. Terada, K. Makino)

Thank you very much

for your kind attention.

Strontium ions as crosslinking agent

Regulation of dextran release rather than
coating polymers on alginate gel beads.
Strontium Ranelate (Protelos): novel antiosteoporosis agent. Sr2+ ions are essential
element in the body. Also the presence of Sr2+
also form alginate gels.
Sr2+-alginate gels were examined for dextran
release behavior.

Effect of metal ions on release

bahavior of dextran
Release behavior of dextran
(MW:1.45x105) from Ca2+-alginate
gels and Sr2+-alginate gels were
compared.

100
90
80
70
60

Sr2+-alginate

50

Ca2+-alginate: a burst release.

Sr2+-alginate:

slow release with

longer release period.

40
30

Ca2+-alginate

20
10
0
0

Release mechanism changed

dramatically with divalent cations.

50

100

150

200

250

300

350

time (min)
beads diameter: 0.50.1mm
meanSD (n=3)

Effect of bead diameter on dextran release

FITC-dex: MW 14.5x104

Onset release time was

shortened by decreasing
gel diameter.
Initial release was
suppressed even for
smaller diameter gels
(see insert).

Cumulative release (%)

For injection route, gel

size should be small.

100
90
80
70
60
50
40
30
20
10
0

0.5mm
(onset: 9min)

25
20
15
10
5
0
0

10

15

2.0mm
(onset: 80min)

25 50 75 100 125 150 175 200

time (min)
meanSD (n=3)