Transcript Synthesis PLGA with enzyme catalyst

PLGA for drug delivery
Huang Juan
Huang Junlian
Saskia Huijser
Rob Duchateau
Introduction

Aliphatic polyesters have been extensively used as important
biodegradable biomaterials for a wide variety of drug delivery
carriers and biomedical devices.

They have biodegradability, versatile mechanical properties and
proven biocompatibility.

Poly(L-lactide)(PLA) and poly(glycolide)(PGA) and poly(lactideco-glycolide)(PLGA) are the most commonly used biodegradable
and biocompatible polymers.
Synthesis of PLGA
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1.Melt polycondensation: step growth
O
O
OH
O
*
Ti(OBu)4
+
HO
Lactic acid
1500C-1800C
HO
OH
Glycolic acid
O
O
n*
m
+ H2O
O
PLGA: poly(lactic acid-co-glycolic acid)
Synthesis of PLGA

2. Ring opening polymerization: chain growth
O
H3C
O
O
O
O
a
a
O
O
CH3
O
O
PS lipase / b SnOct2
100 0C, 7 d / b 180 0C, 6 h
C
O
O
CH
C
CH3
O
CH3
O
CH
C
O
O
CH2
CH2
C
O
n
Lactide
Glycolide
a) Enzyme catalyst
b) Metal catalyst
PLGA: poly(lactide-co-glycolide)
m
Enzymatic polymerization of PLGA
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
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An increase in interest in enzyme-catalyzed organic
reactions
Several advantages:
Catalysis under mild reaction conditions (Temperature, pH,
Pressure)
Nontoxic natural catalyst
Have the ability to be used in bulk reaction media avoiding
organic solvents
Several disadvantages:
Long reaction time
Low molecular weight
Enzyme of lipase PS
Red site: Histidine
Yellow site: Aspartic acid
Green site: Serine
H2N
HO
O
OH
Serine
Postulated Mechanism
O
O
O
R
+ E
Lipase
OH
Lipase-cyclic
compound
Complex
H
O
EM + R'OH
HORCOR'
+
E
OH
(R'=H, Akyl)
Propagation
EM + H
O
O
ORC
n
OR'
H
ORC
C
O
E
Acyl-Enzyme Intermediate
(Enzyme-Activated Monomer,EM)
Cyclic ester
Initiation
R
n+1
OR' + E OH
Results and discussion
The results of PLLA and PGA with/without lipase
Sample
Enzyme
Time
(d)
Conversion
(%)
Mw
(kDa)
PDI
PLLA
PS
7
92
15.0
1.7
PLLA
PS-DI
7
97
9.2
2.2
PLLA
-
7
10
-
-
PGA
PS
2
96
13.0
2.4
PGA
PS-DI
2
100
9.1
1.7
PGA
-
2
0
-
-
Reaction in bulk in 100 0C and using 8 wt % lipase
PLGA prepared by lipase at 100 0C for 7 d
Entry
Samples
Feed
ratio
(L/G)
1a
PLLGA
2
Polymer
ratio
(L/G)
Enzyme
L
Conv
.(%)
G
Conv.
(%)
Mw
(kDa)
PDI
Tg
0
( C)
ΔTg
(0C)
90/10
88/12
PS
89
100
13.9
1.9
50.2
3.6
PLLGA
80/20
77/23
PS
94
100
11.7
1.8
47.5
3.4
3
PLLGA
70/30
70/30
PS
96
100
14.6
1.9
46.4
3.8
4
PLLGA
90/10
82/18
PS-DI
74
100
6.4
1.4
45.7
4.2
5
PLLGA
80/20
69/31
PS-DI
76
100
8.7
1.5
44.7
4.0
6
PLLGA
70/30
64/36
PS-DI
86
100
7.9
1.6
44.0
3.9
7
PDLLGA
80/20
75/25
PS
85
100
10.5
2.4
36.3
5.4
8
PDLLGA
80/20
58/42
PS-DI
78
100
7.4
1.6
35.7
5.0
9b
PLLGA
80/20
79/21
PS
97
100
17.8
2.2
48.3
3.9
10c
PLLGA
80/20
58/42
-
19
57
1.9
1.3
n.d.
n.d.
PLLGA L/G=80/20 using 8 wt% lipase PS at 100 0C
10000
5.0
4.5
100
80
80
60
60
4.0
6000
3.0
Mw
4000
2.0
1.5
2000
1.0
Mw/Mn
2.5
40
40
Lactide
Glycolide
Lactide content (%)
20
20
0.5
0
0.0
0
2
4
Day
6
8
0
0
0
2
4
6
Time (d)
The decrease in polymerization rate may be due to the low concentration
of monomers and the high viscosity of the system.
8
Lactide content (%)
3.5
Monomer conversion (%)
8000
100
PLLGA L/G=80/20 using 8 wt% lipase PS-DI at 100 0C
4.0
6000
5000
3.0
Mw
3000
1.5
2000
1.0
1000
0.5
0
0.0
0
2
4
Day
6
8
Mw/Mn
2.0
110
100
100
90
90
80
80
70
70
60
60
50
50
40
40
Lactide
Glycolide
Lactide content
30
30
20
20
10
10
0
0
0
2
4
6
Time (d)
Both Mw and polydispersity increase during the reaction time. The reaction
rate of glycolide is faster than lactide.
8
Lactide content (%)
2.5
4000
Monomer conversion (%)
3.5
110
8
1. GLGGGG or GGGGLG
2. LGGGLG or GLGGL
3. GGGGG
4. GGGGL + LGGGG
5. LLGGG + GGGLL
6. LLGLL + GLGLL + LLGLG
7. GGGLG + GLGGG
8. Lactydyl region
3
4
1 2
5.30
5.20
5.10
5.00
5
6
4.90
7
4.80
ppm (t1)
1H
NMR spectrum (400 MHz, DMSO-d6) of PLLGA (with 8 wt%
lipase PS at 100 0C for 7 d)
LL
a
LG
b
GG
GL
LL
LG
169.50
GG
169.00
168.50
168.00
167.50
167.00
GL
166.50
ppm (f1)
13C{1H}
NMR spectra (125MHz, CDCl3) of PLLGA (carbonyl region),
a) PLLGA with lipase PS-DI at 100 0C for 7 d. b) PLLGA with lipase
PS at 100 0C for 7 d
LL = (ILL+ILG)/ILG
LG = (IGG+IGL)/IGL
nG
nL

1

LG
H NMR 
LL

13

C NMR
(nL and nG are lactide and glycolide molar fraction in copolymers respectively )
13C{1H}
NMR sequence analysis of PLLGA copolymers
Entry
Glycolide (%)
in polymer [nL/(nL+nG)]
LL
LG
LG/(LG+LL)
(%)
a
31
7.7
3.4
30.6
b
23
10.1
3.2
24.1
A random copolymer would have an average glycolyl sequence
length, LG equal to 2.
MALDI-ToF MS spectra of PLLGA with lipase PS at 100 0C.
Voyager Spec #1[BP = 1214.6, 773]
174.3
100
90
80
% Intensity
70
60
50
40
30
Voyager Spec #1[BP = 1214.6, 773]
20
10
1783.7300
100
0
1500
1933
2366
2799
132.0
0
3665
3232
Mass (m/z )
90
80
1784.7348
1785.7203
% In ten sity
70
1825.6414
1828.6338
60
1827.6826
1797.6875
50
40
1814.6576
1798.6515
30
1809.6728
1787.7305
1777.9642
1826.6705
1813.6666
1786.7088
1781.7152
1821.6534
1823.6369
1829.6363
1815.6427
1789.9164
20
10
0
1773.0
1785.2
1797.4
1809.6
1821.8
0
1834.0
Mass (m/z)
Mass (m/z) = Mend group + mMla + nMga + MK+ (where Mend group = 18 or 0,
Mla = 72, Mga = 58, MK+ = 39)
Main ion series determined by MALDI-ToF spectrum of PLLGA using lipase
Series A
Series B
m
n
m
n
m
n
1783
-
-
17
8
21
4
1785
24
0
13
13
17
9
1797
0
30
18
7
22
3
1799
-
-
14
12
18
8
1811
1
29
19
6
23
2
1813
-
-
15
11
19
7
1821
10
18
-
-
3
27
1825
2
28
20
5
24
1
1827
-
-
16
10
20
6
Series A
Series B
Series C
O
O
H
K
O
O
Series C
Mass
(m/z)
n OH,
m
O
K+
O
O
O
n OH,
m
+
K
K+
O
O
m
n
O
O
PLGA prepared by lipase at 130 0C for 7 d
Entry
Samples
Feed
ratio
(L/G)
1a
PLLGA
90/10
90/10
PS
98
100
19.8
2.3
46.9
4.0
2
PLLGA
80/20
79/21
PS
96
100
20.2
2.7
45.6
3.5
3
PLLGA
70/30
69/31
PS
97
100
18.7
4.6
36.2
6.2
4
PLLGA
90/10
89/11
PS-DI
99
100
11.9
1.5
43.3
4.9
5
PLLGA
80/20
79/21
PS-DI
97
100
11.2
1.6
45.6
3.5
6
PLLGA
70/30
69/31
PS-DI
97
100
10.2
1.6
42.7
4.0
7
PDLLGA
80/20
79/21
PS
97
100
11.5
3.1
43.7
3.8
8
PDLLGA
80/20
79/21
PS-DI
97
100
11.6
1.7
39.2
4.8
9b
PLLGA
80/20
72/28
-
74
100
7.7
1.3
42.9
4.4
Polymer
ratio
(L/G)
Enzyme
L
Conv.
(%)
G
Conv.
(%)
Mw
(kDa)
PDI
Tg
(0C)
ΔTg
(0C)
PLLGA L/G=80/20 using
PLLGA L/G=80/20
8 wt% lipase PS at 1300C
without catalyst at 1300C
6.0
6
7000
5.5
20000
6000
5.0
4.5
5000
15000
4.0
4
2.5
Mw
10000
4000
Mw/Mn
3.0
Mw/Mn
Mw
3.5
3000
2.0
1.5
5000
2
2000
1.0
0.5
0
0.0
0
2
4
Day
6
8
1000
0
0
0
4
2
Day
Mw decreases after the second day. High temperature increases chain
depolymerization. Lipase PS may be denatured at this temperature.
6
PLLGA L/G=80/20 using
8 wt% lipase PS at 1300C
PLLGA L/G=80/20
without catalyst at 1300C
110
100
100
70
100
Lactide
Glycolide
Lactide content
40
40
20
20
60
80
50
70
40
60
50
30
40
20
30
Lactide
Glycolide
Lactide content
20
10
10
0
0
0
0
0
2
4
Time (d)
6
8
-10
-10
0
2
4
Time (d)
High temperature at 1300C increases the polymerization rate.
6
8
Lactide content (%)
60
60
Monomer conversion (%)
80
80
Lactide content (%)
Monomer conversion (%)
90
4.Conclusion
 Lipase PS works as catalyst to synthesize of PLGA and the
conversion gets to 96%.
 Transesterfication has occurred during the reaction.
 PLGA copolymers obtained by lipase PS and lipase PS-DI at
100 0C are block copolymers.
 A higher temperature increases the polymerization rate but
also increases the depolymerization rate.
 The PLGA copolymers from lipase might contain both linear
and cyclic chains.
Acknowledgement

Prof. J.L. Huang
Ir. S. Huijser
Dr. R. Sablong
Dr. R. Duchateau
Prof. C.E. Koning
Dr. F.G. Karssenberg
Prof. P. J. Lemstra

Everybody who contributed to my project
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Thank you for your attention !