Transcript Hydrogels of Hyaluronic Acid Functionalized with

Preparation of Methacryloylb-Cyclodextrin Monomer for Hydrogel
Functionalization
Scott A. Zawko1, Christine E. Schmidt1,2
1Department
of Chemical Engineering
2Department
of Biomedical Engineering
The University of Texas at Austin, Austin, TX
Composite Hydrogel Material
Hyaluronic Acid
Cyclodextrin
Large molecular weight
Host-Guest Inclusion
Complexes
High water content
Increase solubilization
High porosity
Decrease crystallization
Biocompatible
Increase stability
Biodegradable
OH
O
O
OH
HOOC
O
HO
O
OH
HO
O
O
HO
HO
O
O
OH
OH
OH
OH
HO
OH O
O
HO
O
OH
O
OH
NHCOCH3
n
MW = 5x105 – 2x106
O
OH
HO
OH
O
HO
HO
O
O
O HO
O
7
OH HO
O
HO
O
OH
HO
OH
Goal:
Ligands
Create a hyaluronic acid hydrogel that binds and
retains small molecules
Empty Gel
Strategy:
Bound Ligands
Design a cyclodextrin monomer that
binds and retains small molecules
Released Ligands
Design a monomer that binds and
Strategy:
retains small molecules
OH
HO
O
O
HO
O
O
OH
HO
OH
OH
HO
OH O
O
HO
O
OH
Hydrophobic
Interior
O
OH
OH
HO
OH
O
HO
HO
O
O HO
O
OH HO
O
HO
O
OH
g-cyclodextrin (n=8)
b-cyclodextrin (n=7)
a-cyclodextrin (n=6)
HO
OH
Hydrophilic
Exterior
Inclusion Complexes
Ka ~ 105 M-1
+
Adamantane
I.D. = 7.8 Å
O
O
HO
+
C
CH3
CH3
C H 2O H
HO
Ka ~ 103 M-1
CH3
O
O
Hydrocortisone
C
CH3
C H 2O H
Marketed Drug-Cyclodextrin Complexes
a
C yc lo d e x trin
N u m b e r o f A p p ro ve d D ru g s
a -c yc lo d e x trin
3
b -c yc lo d e x trin
10
h yd ro x yp ro p yl-b -c yc lo d e x trin
5
s u lfo b u tyle th e r-b -c yc lo d e x trin
2
m e th yl-b -c yc lo d e x trin
2
a
A p p ro ve d in e ith e r U S , E u ro p e , o r J a p a n
O
HO
C
CH3
C H 2O H
CH3
O
Adapted from Davis M.E. and Brewster M.E. (2004) Nat Rev Drug Discov
Hyaluronic acid
functionalized with MAbCD
Composite Hydrogel:
OH
O
O
HO
O
OH
HOOC
O
O
HO
O
HO
O
OH
OH
HO
OH O
+
O
HO
O
OH
O
OH
OH
NHCOCH3
OH
O
OH
HO
HO
OH
O
HO
HO
3500
I 2959
UV Light
O HO
O
OH HO
O
O
HO
Hyaluronic acid
(HA)
O
OH
Hyaluronic Acid + bCD
Hydrogel
bCD
GMHA Synthesis
O
OH
HOOC
O
O
+
O
HO
Leach, J.B. et al. (2003) Biotechnol Bioeng
O
OH
HO
O
O
NHCOCH3
GMHA
TEA
Synthesis of Methacryloyl-b-Cyclodextrin
M e th a crylic A n h yd rid e
OH
OR
O
O
NaO H, pH 8
O
O
n
HO
n
OH
RO
R = H or
O
n = 6 , a -cy clo de x trin
C
n = 7 , b -cy clo de x trin
OR
O
Possible steric
hindrance
HO
C
CH3
C H 2O H
O
O
O
CH3
O
O
O
O
Characterization: MALDI-TOF mass spectrometry
C ode
N am e
CD
E q u iv a le n ts
of M A
A v e ra g e
DS
F ra c tio n o f
DS 0
2 3
MAbCD4
M AaCD4
a
4 .2
2 .6
0 .0 4
M AaCD6
a
6 .4
3 .4
0 .0 2
M AaCD9
a
9 .6
4 .8
0 .0 0
1
5
0
M AbCD4
b
4 .7
3 .1
0 .0 3
M AbCD7
b
7 .1
4 .2
0 .0 1
M Ab C D 10
b
1 0 .6
5 .2
0 .0 0
C D = c yclo d ex trin.
M A = m ethacrylic a nh yd rid e.
D S = d egree o f sub stitutio n (m o les o f m ethacrylo yl gro up s p er m o le o f C D ).
4
6
Characterization: 1H NMR (solvent: D2O)
Impurities
Impurities
MAbCD
*
6.0
5.0
4.0
3.0
2.0
ppm (t1)
OH
O
O
HO
bCD
OH
*
6.0
ppm (t1)
5.0
4.0
3.0
2.0
*
7
8
18
MAbCD
% Weight
16
7
6
14
DS
12
5
10
4
8
3
6
2
4
2
1
0
0
1
6
11
16
21
26
31
Fraction #
30
8
7
6
20
DS
5
15
4
3
10
2
5
1
0
0
1
6
11
16
21
Fraction #
26
31
Degree of
Substiution
MAaCD
25
% Weight
Water:ACN gradient
98:2 to 45:55
0.8 mL/min
20
Degree of
Substiution
RP-HPLC
Purification
Characterization: 1H NMR (solvent: D2O)
OR
O
O
O
RO
OR
R = H or
*
1
C
2
7
2
1
2
MAbCD
*
Impurities
Crude MAbCD
bCD
6.0
ppm (t1)
5.0
4.0
3.0
2.0
Characterization: MALDI-TOF mass spectrometry
Crude MAbCD
Crude MAaCD
2 3
DS = 3.1
5
1
1
5
0
7
3
DS = 3.2
Pure MAaCD
4
DS = 3.6
6
3
2
4
5
1
5
1
6
0
6
2
4
DS = 3.4
4
Pure MAbCD
3
2
Quantitation of MAbCD-Hydrocortisone Binding
Hydrocortisone
[H y d ro c o rtis o n e ] (m M )
CD (mM):
0
1
3
5
12
MAbCD: 1400 M-1
10
Ka 
8
[ CD : L ]
[ CD ][ L ]
6
bCD: 5380 M-1
4
Ka 
2
0
0
5
10
[C y c lo d e x trin ] (m M )
15
0
slope
(1  slope )[ L ] 0
20
I II III
18
8
IV
7
% Weight
16
6
14
12
5
10
4
8
3
6
Degree of
Substiution
Fractionation of
MAbCD
2
4
2
1
0
0
1
6
11
16
21
26
31
Fraction #
1
2
2 3
4
5
6
3
4
I
DS = 1.5
II
DS = 2.5
III
DS = 3.7
IV
DS = 5.2
6
[H y d ro c o r tis o n e ] (m M )
DS Dependent
Solubility
5
*
* p < 0.05
4
3
2
1
0
bCD
MAbCD4
1 .5
2 .5
3 .7
5 .2
Average Degree of Substitution
1
2
2 3
4
5
6
3
4
I
DS = 1.5
II
DS = 2.5
III
DS = 3.7
IV
DS = 5.2
Hyaluronic acid
functionalized with MAbCD
Composite Hydrogel:
OH
O
O
HO
O
OH
HOOC
O
HO
O
OH
HO
O
O
O
OH
HO
OH
OH O
+
O
HO
O
OH
O
OH
OH
NHCOCH3
HO
OH
O
HO
HO
3500
O HO
O
O
I 2959
UV Light
OH HO
O
HO
Hyaluronic acid
(HA)
OH
HO
bCD
O
OH
Hyaluronic Acid + bCD
Hydrogel
Measure amount of
uncrosslinked MAbCD
Phenolphthalein Assay:
Makela M. et al. (1987) J. Biochem. Bioph. Meth.
O
Ka
O
+
O
O
HO
OH
Phenolphthalein
HO
OH
0 .4 0
0 .3 6
ABS
(550 nm)
50
MAbCD
40
MAbCD, gel
(% w/w)
0 .3 2
bCD
0 .2 8
30
20
0 .2 4
10
0 .2 0
0
0
50
100
[cyclodextrin] (mM)
150
0
10
20
30
40
MAbCD, gelation soln
(% w/w)
50
GMHA-MAbCD Hydrogels: Hydrocortisone Loading
Gel
O
C
CH3
HO
C H 2O H
20 mg/mL GMHA
CH3
+
0, 0.5, 1.0 or 1.5 mg/mL
MAbCD
O
0 .5
0 .4
Hydrocortisone
(mmol)
0 .3
1 HC : 4.6 MAbCD
0 .2
0 .1
0 .0
0 .0
0 .2
0 .4
0 .6
0 .8
MAbCD (mmol)
1 .0
1 .2
Conclusions
Cyclodextrin
Hyaluronic Acid
OH
O
HOOC
O
HO
O
OH
HO
O
O
HO
O
OH
HO
O
O
OH
OH
OH
HO
OH O
OH
O
HO
O
OH
O
OH
NHCOCH3
O
OH
HO
n
MW = 5x105 – 2x106
Composite
GMHA-MAbCD
OH
O
HO
HO
O
O
O HO
O
OH HO
O
HO
O
7
OH
HO
OH
Photocrosslinked Gel
Inclusion complexes
Loaded with Hydrocortisone
Acknowledgements
Dr. Klaus Linse Institute for Cellular and Molecular Biology, UT Austin
Steve Sorey Nuclear Magnetic Resonance Laboratory, UT Austin
Curt Deister, Nathalie Guimard, Quan Truong Schmidt Lab, UT Austin
Funding Agencies
NSF BES – 021744
NSF BES – 0500969
http://www.bme.utexas.edu/faculty/schmidt