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ARTICLE IN PRESS
Contents
1. Introduc
2. Some ba
3. Synthesis
3.1. Ze
3.2. En
3.3. Ba
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Biomaterials 26 (2005) 3771–3782
www.elsevier.com/locate/biomaterials
Review
Chemosynthesis of bioresorbable poly(g-butyrolactone)by ring-opening polymerisation: a review
Tim Moorea,b,�, Raju Adhikaria,c, Pathiraja Gunatillakea,c
aMolecular Science, CSIRO, Bayview Avenue, Clayton South MDC 3169, AustraliabSchool of Engineering and Science, Swinburne University of Technology, John Street, Hawthorn, Vic. 3122, Australia
cPolyNovo Biomaterials Pty Ltd, Bayview Avenue, Clayton South MDC 3169, Australia
Received 4 August 2004; accepted 2 October 2004
Available online 11 November 2004
Abstract
Recent advances in the synthesis of poly(g-butyrolactone) have yielded homopolymers of up to 50,000Mw from the low-cost
monomer g-butyrolactone. This monomer has for the better part of a century been thought impossible to polymerise. Poly(g-butyrolactone) displays properties that are ideal for tissue-engineering applications and the bacterially derived equivalent, poly(4-
hydroxybutyrate) (P4HB), has been evaluated for such uses. The glass transition temperature (�48 to �51 1C), melting point
(53–60 1C), tensile strength (50MPa), Young’s modulus (70MPa) and elongation at break (1000%) of P4HB make it a very useful
biomaterial. Poly(g-butyrolactone) degrades to give g-hydroxybutyric acid which is a naturally occurring metabolite in the body and
it has been shown to be bioresorbable.
Investigation into the synthesis of poly(g-butyrolactone) has recently produced homo-oligomeric diols 400–1000Mw that are
suitable for reacting with diisocyanates to form polyurethanes. Biodegradable polyurethanes made from diols of polyglycolide
(PGA) and poly(e-caprolactone) (PCL) have the disadvantage of high glass transition and slow degradation, respectively. Poly(g-butyrolactone) can be thought of as being the missing link in the biodegradable polyester family immediately between PGA and
PCL and displaying intermediate properties.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Polyhydroxybutyric acid; Polymerisation; Scaffold; Degradation
tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3772
ckground nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3772
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3773
olites and clay as catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3776
zymatic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3776
cterial/microbial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3776
e front matter r 2004 Elsevier Ltd. All rights reserved.
omaterials.2004.10.002
ing author. School of Engineering and Science, Swinburne University of Technology, John Street, Hawthorn, Vic. 3122, Australia.
5 2459.
esses: [email protected], [email protected] (T. Moore).
ARTICLE IN PRESS
Table 1
Comparison of some reported thermal and mechanical properties of
P3HB and P4HB
Polymer Tg (1C) Tm (1C) Tensile
strength
(MPa)
Elongation
%
Ref.
P3HB 1–5.3 150–180 36 3 [4–6]
P4HB �48 to
�51
53–60 50 1000 [4,7–9]
Table 2
Structures and nomenclature of some common lactones
4. Mechanism of ring-opening polymerisation of
g-butyrolactone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3777
5. Degradation of poly(4-hydroxybutyrate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3777
6. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3777
6.1. Endogenous levels of 4-hydroxybutyrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3777
6.2. Exogenous 4-hydroxybutyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3778
6.3. Genotoxicity studies of g-butyrolactone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779
T. Moore et al. / Biomaterials 26 (2005) 3771–37823772
1. Introduction
Degradable polyesters have found a wide range ofuses in medical applications such as absorbable suturesand tissue-engineering scaffolds. The ester linkage issusceptible to hydrolysis and has been shown to degradeboth hydrolytically and enzymatically. The degradationproducts of the biodegradable polyesters are typicallyhydroxy acids, such as glycolic acid and lactic acid thatare generally recognized as being non-toxic. For thesereasons there has been a large volume of research intothe biodegradable polyester family in the past fewdecades.
Recently, poly(4-hydroxybutyrate) (P4HB) has at-tracted the interest of researchers for use as abiodegradable polymer. It has been evaluated asscaffolds [1–3] in combination with other degradablepolyesters for tissue engineering of cardiovascular tissue.P4HB displays similar physical properties to poly(e-caprolactone) (PCL), while degrading in a shorterperiod of time [4] which makes it a valuable additionto the tissue-engineers repertoire.
This paper reviews aspects of chemosynthesis by ring-opening polymerisation of g-butyrolactone (g-BL) aswell as degradation and toxicity of P4HB.
2. Some background nomenclature
There are two BL isomers (Fig. 1): g-BL, which is thesubject of this paper, and b-butyrolactone (b-BL), which
O OH
O
Hn
O
O
O
O
O O
O
HH n
Fig. 1. Polymerisation of g-BL (left) to P4HB and b-BL (right) to
P3HB.
is more easily polymerised by ring opening due toincreased ring strain.
Many authors in the past have referred to P3HB assimply polyhydroxybutyrate (PHB) since P4HB was notknown, and this nomenclature is often still in use. Themechanical and thermal properties of the two materialsare very different (Table 1) where P4HB is straight chainand P3HB has a methyl side-group (Table 2). Note thatP3HB contains a chiral centre (as does lactic acid) andthe properties of the polymer depend upon the tacticity.
In the literature, the nomenclature ‘lactone’ has oftenbeen used interchangeably with ‘cyclic ester’ and theyhave been named based on the number of members inthe ring as well as the number of carbons in themonomer (Table 2).
Polymers in general are usually named after thestarting monomers. For example, ‘‘polyglycolide’’
Lactone Structure Lactone Structure
b-Propiolactone
O
O b-Butyrolactone
O
O
g-ButyrolactoneO
O b-Valerolactone
O
O
d-ValerolactoneO
O g-ValerolactoneO
O
e-CaprolactoneO
O
ARTICLE IN PRESS
nO
rigin
Pro
per
ties
Ref
.
Chem
osy
nth
etic
Hard
,britt
leand
inso
luble
in
most
solv
ents
[15–20]
Chem
osy
nth
etic
Toxic
deg
radation
pro
duct
s[8
,10,1
8–20]
Bact
eria
lSoft,pla
stic
ther
mally
pro
cess
ible
[4,9
]
Bact
eria
lor
chem
osy
nth
etic
Soft,pla
stic
sim
ilar
toPeC
L[1
8,2
1]
Chem
osy
nth
etic
Soft,pla
stic
and
hydro
phobic
[22–24]
T. Moore et al. / Biomaterials 26 (2005) 3771–3782 3773
(PGA) is synthesised by ring opening the glycolide cyclicdimer and it can be distinguished from ‘‘poly(glycolicacid)’’ which is polymerised by condensation of glycolicacid. Unfortunately, this has not always been the casewith lactones where the polymer has sometimes beennamed after the repeat unit rather than the startingmonomer, as in the example of ‘‘poly(3-hydroxybuty-rate)’’ which was made by ring opening of b-BL [5,11].Another reference named the same polymer as ‘‘poly(b-hydroxybutyrate)’’ [12], while some references name thepolymer after the lactone: b-BL was polymerised to givepoly-b-BL, ‘‘poly(b-butyrolactone)’’ [13].
b-Propiolactone is a relatively potent carcinogen—ithas a TD50 of 1.16 mg/kg body weight/day [14]. Due tothe carcinogenicity of b-propiolactone, poly(b-propio-lactone) has not usually been considered for use inbiomedical applications. Bearing this in mind, one seesan obvious gap in properties of degradable polyesters(Table 3) where P3PL is unsuitable and P4HB has beenunpolymerisable, which leaves the very rigid and fast-degrading PGA and the slow-degrading Poly(d-Valer-olactone) (PVL) and PCL (Table 4).
Table
3
Mec
hanic
aland
ther
malpro
per
ties
ofso
me
linea
raliphatic
deg
radable
poly
este
rs
mPoly
este
rM
elting
poin
t(1
C)
Tg
(1C
)T
ensile
stre
ngth
(MPa)
Young’s
modulu
s(M
Pa)
Elo
ngatio
at
bre
ak
%
1Poly
(a-g
lyco
lic
aci
d)
(PaG
A)
223–233
46.5
100
6300
1.5
2Poly
(b-h
ydro
xy
pro
pio
nate
)(PbP
A)
77
�19
103
1590
500–600
3Poly
(g-h
ydro
xybuty
rate
)
(PgH
B)
53
�51
50
70
1000
4Poly
(d-h
ydro
xyvale
rate
)
(PdH
V)
57
�55
12.5
570
150–200
5Poly
(e-c
apro
lact
one)
(PeC
L)
60
�65
37
216
746
HO
C H2
OH
O
mn
3. Synthesis
Of all the degradable polyesters, P4HB (or poly(g-butyrolactone)) has attracted the least attention and hasarguably been the most misunderstood due to amisconception g-BL cannot undergo ring-opening poly-merisation. Initial research into the polymerisation of g-BL by Carothers et al. [29] stated in 1932: ‘‘Thus, wehave heated samples of pure g-butyrolactone both withand without catalysts (zinc chloride, potassium carbo-nate) at 801C for 12 months; none of the samplesshowed any detectable increase in viscosity.’’ A conclu-sion was drawn from this that g-BL was not able toundergo ring-opening polymerisation at all and thisconclusion has since been explained thermodynamically[30–34]. Despite this failure to polymerise, there havebeen numerous groups [29,35,36] that have publishedattempts to homopolymerise g-BL without successand one would assume that many more groups mighthave been reluctant to publish negative results consider-ing it had already been reported as being unable topolymerise.
In 1951, g-BL was shown to ring open to formoligomers (a degree of polymerisation of 2–3) withdiethoxy terminal groups [37]. This was a side-productof the reaction and was not a desired product; however,it showed for the first time that g-BL could homo-polymerise, or more correctly oligomerise.
Despite this, it was still thought [38] that g-BL wasunable to ring-open polymerise at all and it was not until1966 that this was for the first time purposely shown tobe not correct [39]. In that case, extreme conditions were
ARTICLE IN PRESS
Table
4
Typic
alm
echanic
aland
ther
malpro
per
ties
oftw
oaliphatic
deg
radable
poly
este
rsth
athave
met
hylside-
gro
ups
Poly
este
rM
elting
poin
t
(1C
)
Tg
(1C
)T
ensile
stre
ngth
(MPa)
Young’s
modulu
s
(MPa)
Elo
ngation
at
bre
ak
%
Origin
Pro
per
ties
Ref
.
PL
LA
,poly
-L-lact
ide
180.7
–192.5
56.6
–66.8
64.3
–77
619.7
–2380
1.6
2–8
Chem
osy
nth
etic
Hard
and
britt
le[2
5,2
6]
PbH
B,poly
(b-
hydro
xybuty
rate
)
174.3
–177
4–5.3
28–36
1688–3670
1–7.5
Bact
eria
lor
chem
osy
nth
etic
Hard
and
britt
le[4
,7,2
6–28]
T. Moore et al. / Biomaterials 26 (2005) 3771–37823774
used to achieve polymerisation (20,000 atm and 165 1C)and homopolymers of between 1200 and 3350Mw wereformed (the molecular weight was measured using avapour pressure osmometer).
Kricheldorf et al. [40] stated in 1985: ‘‘Concerningcopolymerizations of g-butyrolactone, it is noteworthythat this monomer for thermodynamic reasons cannotbe homopolymerised at temperatures above 50 1C.’’ In1991, Jedlinski et al. [41] also stated that g-BL cannothomopolymerise for thermodynamic reasons. As re-cently as 2003, g-BL has been referred to in the literatureas ‘‘nonhomopolymerizable’’ [42]. While there is somemerit to these statements, they neglect to take intoaccount the effect of catalysts and pressure that havefacilitated the formation of quite high molecular weightat temperatures above 50 1C. The reason these authorsstated that it cannot form a high molecular weighthomopolymer is that it has a very small ring strain, sosmall that DGp (Gibbs free energy of polymerisation) ispositive [43]. Simply put, this means that the ester in thelactone ring is less likely or as likely to break and joinonto the polymer chain (due to its stability) than theester in the free polymer chain is likely to transesterify orundergo ring closure under normal conditions. Thethermodynamic parameters involved in the polymerisa-tion of g-BL to P4HB at both normal pressures [33] andin the super-cooled state under high pressures [44] havebeen investigated.
g-BL does not undergo ring-opening polymerisationas easily as b-BL does because of the aforementionedsmall ring strain [43]. High molecular weight poly(3-hydroxybutyrate), e.g. Mw 430,000 [45] and 580,000 [6],has been made by ring-opening polymerisation withoutmuch difficulty since the four-membered ring has agreater strain than the five-membered ring.
While it has been calculated [46] to be thermodyna-mically impossible to chemically synthesise a highmolecular weight homopolymer under normal condi-tions, numerous papers have shown that g-BL can becopolymerised with other lactones and hydroxy acids, toform copolyesters [5,34,46,47,48] (Table 5) and can evenform homopolymers of low molecular weight [38,49,50](Table 6).
The molecular weight of homopolymers of g-BLachieved by ring opening that have been reported todate are low, e.g. 200 Da (a degree of polymerisation of2.4 was published [49] based on 1HNMR data).
Mn (by GPC) of 800 Da has been achieved [57] byusing a lipase from Pseudomonas sp. to catalyse thering-opening reaction. This reaction was carried outwith 0.10 mmol (0.0086 g) of g-BL and 0.040 g of lipaseat 45 1C over a period of 20 days and yet achieved onlyan 8% yield and Mw/Mn=2.23 after precipitation.Precipitation tends to reduce Mw/Mn and increase Mwby removing some of the low molecular weightoligomers.
ARTICLE IN PRESS
Table 5
Some reported successful attempts to copolymerise g-BL by ring-opening polymerisation
Year Co-monomer Temperature (1C) Time Mn Max % g-BL in product Yield % Ref.
1964 b-PL 50 20 days NA Feed 98:2, BL:PL 0.2 [51]
1964 BCMO 0 3–35 h NA 89.7% 6.0 [52]
1964 b-PL 30 21 days NA Feed 7:3, BL:PL 43 [52]
1969 BCMO 20 20 h NA 50% 17.4 [53]
1970 BCMO 25 6.4 h NA 36% 7.9 [54]
1985 GA 60 44 h NA 26% 1.4 [40]
1989 L-LA 200 20 h 640 19% NA [36]
1990 GA 200 7 h 1500 16% NA [48]
1995 b-BL 100 4 h 2700 35% 13 [5]
1996 L-lactide 140 4 days 14,600 17% 6 [55]
1997 b-BL 25 7 days 1800 56% 24 [56]
1998 e-CL 140 4 days 29,500 16% 16 [47]
1998 d-VL 140 4 days 18,600 15% 12 [47]
1998 b-PL 140 4 days 1600 23% 9 [47]
1998 GA 140 4 days NA 26% 26 [47]
1998 e-CL 45 20 days 2900 5% 45 [57]
1999 e-CL 25 2 h 57,000 22% 31 [34]
2002 d-VL 25 24 h DP=4.3 30% 5.7 [49]
2003 e-CL 25 48 h 2880 33% 38 [42]
Table 6
Some reported successful homopolymerisations of g-BL
Year Temperature
(1C)
Time (h) Length of polymer or
oligomer formed
Characterisation of
molecular weight
Yield % Notes Ref.
1951 NA NA Low DP, 2–3 and
more
Mp NA Diethoxy-terminated (side-
product)
[37]
1966 160 4 1200–3350 by
osmometry
IR (not shown) 20 20,000 atm, recrystallised three
times, mp 61–62 1C
[39]
1996 60 430 888–932Mw MALDI-TOF MS 25–42 Lipase catalyst in n-hexane using
methanol as initiator (�2:1 of
lipase to g-BL by mass)
[58]
1998 45 480 Mn 800 (by GPC)
Mw/Mn=2.23
HNMR and CNMR
(not shown)
8 8.9 mg g-BL to 40mg of lipase [57]
1999 144 21 400–1000Mn by GPC HNMR and GPC
shown
56 Clay catalyst, initiated with
ethylene glycol and diethylene
glycol
[50]
2000 40–160 10–70 10,000–50,000Mw GPC 9–74 Lewis acid catalyst at very high
pressure. Less than 4 g reactants
[59]
2002 25 3 DP 2.4 (Mn�200) HNMR (not shown) 5.5 Ethanol initiated, clay catalyst
(ion-exchanged montmorillonite)
[49]
2003 180 6 NA (oligomers) None shown 47.2 Not the intended product, zeolite
catalyst
[60]
2003 40–160 5–300 Mn=‘‘5000 or more’’ GPC 5–23 Metal complex catalyst at very
high pressure. Less than 1 g of
reactants
[61]
T. Moore et al. / Biomaterials 26 (2005) 3771–3782 3775
Oligomers of 4HB are useful prepolymers that canundergo chain-linking reactions such as with diisocya-nates to form polyurethanes [50] or other polymers.Many of the attempts to polymerise g-BL have not beencarried out with the intention to yield the diols necessaryfor polyurethane chemistry. The initiators used aretypically unsuitable, e.g. ethanol [49], which gives anethoxy-terminated chain, which prevents further poly-merisation (Fig. 2). One obvious way to circumvent this
problem is to use a difunctional initiator like a diolrather than the monofunctional alcohol, e.g. ethyleneglycol [50] (Fig. 2). This gives a diol that could beused for the synthesis of biodegradable polyurethanes(Table 7).
Bailey et al. [66] in 1976 describe developments insynthesis of alternating poly(ester–ether)s from spiro-ortho-esters, some which include g-BL as a monomer(Fig. 3). These do not form the P4HB homopolymer but
ARTICLE IN PRESST. Moore et al. / Biomaterials 26 (2005) 3771–37823776
are considered biodegradable and useful for biomedicalapplications.
3.1. Zeolites and clay as catalyst
Mesoporous zeolites were shown to be effectivecatalysts for ring-opening polymerisation of d-valero-lactone and e-caprolactone [67]; however, the authorsdid not report attempting g-BL. Another group reportedthat an unexpected oligomerisation of g-BL occurredduring an attempted alkylation over zeolites [60]. Theydid not show the characterisation of these oligomers,however reported a 47% yield of oligomer; the reactiontemperature was 180 1C and reaction time was 6 h. As aresult, zeolite shows potential as a catalyst for g-BL ring-opening polymerisation and has not yet been thoroughlyinvestigated.
CH2
CH2
OHOH
O
O
CH2
CH2
O CH2
CH2
CH2
O H
O
mO
O
CH2
CH2
CH2
OHm
CH3 CH2
O CH2
CH2
CH2
O H
O
m
CH3 CH2
OH
Heat
Heat
Fig. 2. Ring opening of g-BL with ethanol or ethylene glycol.
Table 7
Some reported unsuccessful attempts to polymerise g-BL
Year Catalyst/initiator
1932 Zinc chloride, potassium carbonate and none
1961 14 different organometallic catalysts
1964 BF3.Et2O
1977 Al2/Zn m-oxoisopropoxide
1989 None
1997 Cationic zirconocene dimethyl complexes
1999 Samarium(II) aryloxide complexes
2003 Zwitterionic titanoxanes
2003 Amino isopropoxyl strontium
2003 SmI2/Sm
OO
O
O
O
O+
Fig. 3. Formation of poly(ester–ether) alt
Ion-exchanged montmorillonite clay has also shown arelatively high catalytic conversion of the monomer (e.g.56% yield of 1000Mn P4HB) [50], which is notsurprising considering the similarity between the zeoliteand montmorillonite structures (both are aluminosili-cates).
3.2. Enzymatic
Uyama and Kobayashi [68] showed for the first timethat lactones could be ring-opening polymerised with alipase to form polymers in 1993 when they polymerisede-caprolactone and d-valerolactone to a maximummolecular weight of 7700Mn.
There are not many cases of ring-opening polymer-isation of g-BL using enzymes in the literature to date;however, this may change as lipases have been shown tobe effective catalysts for the polymerisation. However,there is a major disadvantage with all the reportedoligomerisations of g-BL using lipases: they use a largeratio of lipase to g-BL making it quite expensive,especially since enzymes are known to lose their catalyticeffect over time. For example, lipase from Pseudomonas
sp. was used to ring-open g-BL to give 800Mn at 45 1C,480 h, and with a yield of 8% [57]. Unfortunately, thereaction used 40mg lipase to 8.9 mg of g-BL yieldingonly 0.71 mg poly(g-butyrolactone) which makes this anexpensive exercise.
3.3. Bacterial/microbial
It is worth noting that P4HB of high molecularweight has been made using microbes (Table 8). Thefeedstock for the micro-organisms has included g-BL for
Temperature (1C) Time Ref.
80 12 months [29]
0–78 3–72h [62]
30–80 9–30 days [52]
40 NA [35]
200 20 h [36]
25–60 2 h [63]
25 1–5min [34]
20 NA [64]
25–80 70min [65]
25 48 h [42]
* O
O
O *n
ernating copolymer including g-BL.
ARTICLE IN PRESS
Table 8
Four categories of P4HB synthesis and approximate Mw range
achieved
Type Method of synthesis of P4HB
from g-BL
Approximate Mw
range
1 Chemical catalyst at
atmospheric pressure
0–1000
2 Lipase catalyst in vitro 200–900
3 Chemical catalyst at ultra-high
pressure
1300–50,000
4 Micro-organisms 1,000,000
T. Moore et al. / Biomaterials 26 (2005) 3771–3782 3777
P(3HB-co-4HB) synthesis using bacteria (1992) and upto 100% composition of P4HB has been made [7].Thermal degradation of P4HB shows g-BL as a majordegradation product (1994) [69]. Properties of P4HB(bacterial) have been characterised and show it to be astrong flexible polymer (Table 1) [8].
Poly-4-hydroxybutyrate has recently been made by afermentation process using genetically engineered Es-
cherichia coli that is capable of producing up to 50 g ofpoly-4-hydroxybutyrate per litre of fermentation brothin 48 h [4].
4. Mechanism of ring-opening polymerisation of
c-butyrolactone
Ring-opening polymerisation has been carried outusing either cationic catalysts or anionic catalysts. Thegenerally accepted mechanism for the cationic ring-opening polymerisation of g-BL involves coordinationand alignment of the carbonyl oxygen with the metalcentre (e.g. Al) followed by insertion.
g-BL has been used to determine the mechanism ofinitiation of ring-opening polymerisation using a stron-tium-based initiator system [65]. The authors chose g-BLbecause it would react with the initiator but not furtherpolymerise under normal conditions. The results showedthe ring-opening polymerisation followed a coordina-tion–insertion mechanism. The general mechanism forP3HB has also been proposed and is considered tofollow a similar mechanism [107].
Data have been published detailing the relativecatalytic copolymerisation parameters of some commonlactones including g-BL and as one would expect it isunlikely to homopolymerise [108]. Formation of analternating copolymer between g-BL and 3,3-bis(chlor-omethyl)oxacyclobutane at high BL feed content wasexplained by the authors as due to the inability of BL tohomopolymerise [53].
The mechanism can be explained in part by the factthat g-BL is shown to form a one-to-one adduct withcertain initiators including for example yttrium methox-ide [109] through coordination of the g-BL carbonylwith the yttrium metal centre.
5. Degradation of poly(4-hydroxybutyrate)
Nakayama et al. [47] showed that introduction of g-BL units into polyesters resulted in both enhancedbiodegradability and flexibility. Polyesters copoly-merised were: PLLA; PGA; poly-b-propiolactone;poly-d-valerolactone and poly-e-caprolactone.
It has been published that the body absorbs P4HB ina period of 8–52 weeks [4]. It is also stated in the samereference that due to cyclisation of the degradation
product from hydroxy acid to lactone, the degradationproducts are somewhat less acidic than those of PGAand PLA which both have lower pKa values.
P4HB has been tested for degradability [8] in riverwater (containing micro-organisms) at 25 1C and com-pared with other polyesters. P4HB was the polyesterthat degraded at the highest rate (by biological oxygendemand) in the presence of these micro-organisms.
Thermal degradation of P4HB has shown that thepyrolysate contains mainly g-BL and higher oligomersboth cyclic and linear to pentamer [69].
6. Toxicity
When considering toxicity one must take into accountthe biocompatibility of the initial polymer as well as thatof the degradation products. There has been some workpublished regarding the biocompatibility of bacteriallyderived P4HB for tissue engineering [70–75].
The main use of P4HB has been to provide strength tobiodegradable non-woven PGA and PLLA scaffolds bydip coating into a 1% solution of P4HB in tetrahy-drofuran [70,71]. These scaffolds have been reported in anumber of papers to have good biocompatibility andfast degradation. A scaffold for a human pulmonaryconduit was shown to be replaced by living cells in vitro[70]. Ovine mesenchymal stem cells have also beenshown to proliferate on the P4HB-coated PGA scaffolds[73], as have human umbilical cord cells [74], and ovinemyofibroblasts and endothelial cells [75].
P4HB has been said to be not only biocompatible butalso often extremely well-tolerated in vivo [4]. Theauthors also suggest that P4HB implants are unlikely tocause any adverse pharmacological effects due to theirrelatively small sample size, slow release and rapidmetabolism.
6.1. Endogenous levels of 4-hydroxybutyrate
The final degradation products upon hydrolysis of theester linkages of poly(g-butyrolactone) are g-BL and 4-hydroxybutyric acid both of which are essentially the
ARTICLE IN PRESST. Moore et al. / Biomaterials 26 (2005) 3771–37823778
same thing in vivo since in the body, g-BL rapidlyundergoes ring opening catalysed by the enzyme g-lactonase to give 4-hydroxybutyric acid.
It has been known for some time that 4-hydroxybu-tyric acid is endogenous in the human brain [76], butmore recent findings have shown that it can be found ina number of other organs and in blood samples;however, care must be taken in the interpretation ofresults since tissue samples that are not fresh or frozenimmediately after death have been shown to giveincreased readings. Fresh human blood has been foundto contain 0.17–1.51 mg/l of 4-hyroxybutyrate [77];however, some other studies have been unable to detecttraces of 4-hydroxybutyrate in healthy individuals[78–80]. In another study, recoveries greater than100% in samples of blood and urine spiked with 4-hydroxybutyrate have been attributed to endogenousconcentrations [81]. The concentration of 4-hydroxybu-tyrate in autopsy blood has been shown to increase overthe time between death and autopsy [78] and the authorssuggest it is most likely due to enzymatic conversion ofsuccinic acid, g-aminobutyrate and putrescine. Theconcentration of 4-hydroxybutyrate in post-mortemblood stored for 10 days at 4 1C was 6.0674.27 mg/lbut only 4.5573.88 mg/l when stored for the sameperiod frozen at �20 1C [78] and a positive correlationwas found between concentration and post-morteminterval. Human liver samples have also been shownto exhibit the same post-mortem increase [80]. Endo-genous 4-hydroxybutyrate concentrations of �0.25 mg/lhave been found in human urine and have been shownto increase in concentration up to 404% in 6 monthsdepending on storage conditions [82]. Human sigmoidcolon samples explanted from the gastrointestinal tractcontained endogenous 4-hydroxybutyrate at concentra-tions of 2.59–4.19 mg/kg [83].
Succinic semialdehyde dehydrogenase deficiency(SSADH) is a rare hereditary ailment where succinicsemialdehyde is not converted to succinic acid but ratherfollows an alternative pathway to form 4-hydroxybuty-rate [84]. A review of SSADH cases [84], showedelevated cerebrospinal fluid concentrations of 4-hydro-xybutyrate from 65 to 230 times the normal levels(46.774.3 mg/l) in combination with three times thenormal g-aminobutyrate concentration and a lowconcentration of glutamine. Significant behaviouralproblems were evident in 42% of patients, and mostwere said to experience global development delays and50% experienced seizures [84].
6.2. Exogenous 4-hydroxybutyrate
There is a history of human consumption of g-BL andit has been used as a flavouring ingredient [85], as asedative [86], for bodybuilding [87,88], in the treatmentof alcoholism and opiate dependence [89,90]. It is
naturally occurring in some food, has been used as adrug of dependence [87,91–94] and is characterised as a‘date-rape’ drug [95,96]. Hence, there are a number ofreferences detailing the metabolism of g-BL in thehuman body as well as numerous toxicity studies andthey have previously been reviewed [85].
The half-life of g-BL in plasma is less than a minute[85] before conversion to 4-hydroxybutyric acid by g-lactonase, and the maximum concentration of 4-hydro-xybutyrate in plasma occurs 20–60 min after oraladministration [97] before �2–5% is excreted in urine[98], and the majority has been shown to be metabolisedand exhaled as CO2(g) within 150 min [99]. Automobiledrivers in the Netherlands have had readings of 4-hydroxybutyric acid as high as 2000 mg/l in urine and ashigh as 194 mg/l in blood [95]. The reason the drivershad 4-hydroxybutyric acid in their system is because itgives the user a ‘‘high’’ when taken orally. It iscommonly reported that it is abused for its euphoriceffects, as well as for reported properties of increasingmuscle mass and sexual pleasure [95].
When a 25 mg/kg dose of sodium 4-hydroxybutyratewas administered orally to a human volunteer, 4-hydroxybutyrate was detectable in urine at 30 min, washighest at 1 h (30.3mg/l), and was undetectable after 4 h(o2 mg/l) showing that it is rapidly absorbed andeliminated [98]. The dose–response curve for 4-hydro-xybutyrate is purportedly steep, where 40–50 mg/kg cancause somnolence leading to arousable sleep and60–70 mg/kg can cause coma for 1–2 h [97].
There have been a number of studies and reviews thatshow 4-hydroxybutyrate to have therapeutic value in thetreatment of alcoholism [100–105] and opiate depen-dence [90,102]. Doses of 4-hydroxybutyrate have beenshown effective in suppressing the effects of alcoholwithdrawal without serious side-effects [100]. Theauthors speculate that the mechanism of action of 4-hydroxybutyrate is an interference with the release ofdopamine and serotonin which are the main modulatorsof the ethanol reward system [100]. The mechanism hasbeen reviewed in more detail [104] and is thought to beof a substitutional nature.
Bodybuilders have used 4-hydroxybutyrate as a diet-ary supplement since research was published showing itincreases production of certain growth hormones inhealthy humans, yet other research shows it does notbuild muscle mass in rats, dogs or alcoholics [105,106].The recommended daily dosage for bodybuilders is1.4–2.8 g of 4-hydroxybutyrate with some reportedlytaking considerably more [96].
There are reports of people who have displayed anaddiction to 4-hydroxybutyrate or its precursor g-BL[87,91–93]. A typical case involves a 36-year-old manwho imbibed �5 g g-BL every 2 h around the clock for 6months [87]. Upon forced removal from the use of g-BLhe displayed withdrawal symptoms such as tremors,
ARTICLE IN PRESST. Moore et al. / Biomaterials 26 (2005) 3771–3782 3779
sweating, hallucinations and delirium; however, he wasasymptomatic after 3 days treatment.
One must bear in mind that an implanted P4HBscaffold weighing only a few grams would release 4-hydroxybutyrate over a long period and would not havesignificant pharmacological effects. For example, 2 g ofpure P4HB implanted in a 70 kg person which degradedin 2 months would average just under 0.5 mg/kg/day of4-hydroxybutyrate, which is negligible considering en-dogenous concentrations in blood have been measuredto be 0.17–1.51 mg/l [77], endogenous gastrointestinalconcentrations of 2.59–4.19 mg/kg [83], and therapeuticdoses of 50–100 mg/kg/day have been used in thetreatment of alcoholism [100].
6.3. Genotoxicity studies of g-butyrolactone
The Flavour and Extract Manufacturers’ Association(FEMA) Expert Panel has reviewed more than 50genotoxicity studies of g-BL and concluded: ‘‘ythat g-butyrolactone (4-hydroxybutanoic acid lactone) is notmutagenic and that isolated positive results performedin non-standard assays at high solution concentrationsare not compelling evidence of genotoxic potential. Thenegative response in repeated Salmonella mutagenicityassaysysupports the Panel’s conclusion that exposureto 4-hydroxybutanoic acid lactone exhibits little poten-tial for interaction with DNA.’’ [85]
7. Conclusion
Oligomers and co-oligomers containing g-BL haverecently been shown to be chemosynthetically possibleprecursors for biodegradable polyesters and polyur-ethanes, which have many potential uses in the field ofbiomaterials. Bacterially derived poly(4-hydroxybuty-rate) has been evaluated for use in a number ofbiodegradable scaffolds both in vitro and in vivo withpromising results showing it to be a strong, flexible andbiocompatible polymer with a relatively rapid degrada-tion profile. This suggests that this hitherto little-explored branch of the biodegradable polyester familymay provide extremely valuable novel biomaterials fordrug delivery and tissue engineering, thus warrantingfurther investigation.
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