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Cryo-TEM and molecular rotor fluorescence as complementary techniques for the characterization of cationic amphiphilic copolymer microdomains in aqueous medium

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Polym Int 48 :283–287 (1999)
Polymer International
A route to high radioactivity 3H-labelled PLA
Is abelle Dos Santos , Jean-Louis Morgat and Michel Vert*
CRBA URA CNRS 1465 , Univers ity Montpellier 1 , Faculty of Pharmacy , 15 avenue Charles Flahault , 34060 Montpellier Cedex , France
Abstract : In the üeld of degradable and biodegradable polymers, monitoring the fate of degradation
products in complex living systems such as an animal body or the environment, is going to be a major
task with the development of forth-coming regulations. For this, radiolabelling is a powerful tool,
provided that no undesired radioactive leachables lead to artifactual observations. Therefore, a
method to synthesize highly radioactive polylactide polymers has been investigated. 1H Ç 3H isotope
exchange was achieved on L- and DL-lactides using tritium gas and Pd/CaCO as catalyst. Radioactive
lactides with speciüc activities ranging up to 111–148 GBq mmol—1 (3–4 Ci mmol—1) were obtained.
With these lactides, PLA50, PLA96 and PLA100 (M1 = 25 000–60 000) were synthesized with speciüc
radioactivities in the range 29.6–44.4 MBq mg—1 (0.8–1.2 mCi mg—1). It was shown that no more than
0.45% of volatile radioactive compounds were formed within a typical polymer, namely PLA50, which
was allowed to stand in air at 4ÄC for 200 days. PLA50 tablets and thin ülms were allowed to degrade
hydrolytically in an iso-osmolar pH 7.4 phosphate buþ er solution at 37ÄC for several weeks. The
appearance of radioactive leachables in the aqueous medium agreed well with the heterogeneous degradation mechanism previously proposed to account for the behaviour of large-size cold PLA polymers in vitro and in vivo. Data are discussed with respect to the use of radioactive PLA as tracers in
the animal body and in model compost.
( 1999 Society of Chemical Industry
Keywords : lactide ; PLA ; radiolabelling ; tritium ; bioresorption ; degradable polymers
Because of their biocompatibility and sensitivity to
water, poly(a-hydroxy acids) derived from lactic acid
(PLAX in which X is the percentage of L units) have
been proposed as degradable polymers of therapeutic
interest.1 Nowadays, PLA polymers are also being
produced for environmental applications, and are at
the pre-development stage in the üelds of packagings
and ülms.1 The monitoring of the fate of degradation
products in the corresponding living systems
requires special tools. Radioactivity-based methods
have been developed for drugs in pharmacology. It is
likely that regulatory agencies are going to request
similar approaches to validate the use of degradable
and biodegradable polymers in other üelds of applications involving complex living systems. A few data
have been reported in the literature on the use of 14C
to radiolabel degradable and biodegradable polymers ; however, the polymer radioactivity was often
too weak to provide üne and reliable detection.2h4 A
tritiated polylactic acid of very low speciüc activity
has also been described, but no indication of the tritiation method was provided.5
In this paper, we wish to report a method to
radiolabel PLA polymers by exchanging protons for
highly radioactive tritium in the presence of a catalyst, and to validate the method insofar as the
absence of signiücant radiolysis and early release of
radioactive leachables are concerned. The high temperature solid state catalytic isotope exchange
(HSCIE) proposed by Zolotarev and co-workers6h9
was adapted to the case of L- and DL-lactide labellings. For convenience, experimental conditions were
investigated and optimized with deuterium, the other
(non-radioactive) isotope of hydrogen. Optimization
was achieved on the basis of 1H NMR, Raman spectroscopy and mass spectrometry analyses. Various
PLA stereocopolymers derived from these tritiated
monomers, namely PLA50, PLA96 and PLA100,
were synthesized by ring opening polymerization initiated by stannous octoate and by anhydrous zinc
lactate, the two compounds which are currently used
industrially as initiators.
* Corres pondence to : Michel Vert, CRBA URA CNRS 1465, Univers ity Montpellier 1, Faculty of Pharmacy, 15 avenue Charles
Flahault, 34060 Montpellier Cedex, France
(Received 1 June 1998 ; accepted 30 September 1998 )
DL- and L-lactide were obtained from Purac
(Gorinchem, Netherlands) and were recrystallized by
sublimation at 57¡C under vacuum (10~3 bar). 5%
( 1999 Society of Chemical Industry. Polym Int 0959-8103/99/$17.50
I Dos Santos, J-L Morgat, M Vert
Pd/CaCO were purchased from Acros Organics
(Geel, Belgium). Commercial anhydride THF from
Carlo Erba (Milan, Italy) was distilled in the presence of sodium metal before use. Stannous octoate
was purchased from Aldrich (Milwaukee, USA) and
used as received. Zn lactate was obtained from
Zn-DL-lactate, 3H O purchased from Sigma (St
Louis, USA) and dried at 200¡C for 12 h. All the
solvents (acetone, chloroform and ethanol) were of
analytical grade. They were used without further
Polymer recovery
In the three cases, the sealed ýasks were allowed to
stand in an oven thermostatted at 140¡C for the
selected reaction time, after which the polymers were
recovered by dissolution in acetone for PLA50 or in
chloroform for PLA100 and PLA96. The unreacted
lactides and the low molecular weight oligomers were
removed by precipitation in ethanol. The resulting
polymeric materials were cooled with liquid nitrogen
and the residual solvent was evaporated using
dynamic vacuum.
Tritiated DL-lactide and L-lactide
DL-Lactide or L-lactide (50 mg) was introduced into a
3.2 cm diameter round-bottomed ýask. THF (1 cm3)
and 250 mg of 5% Pd/CaCO were added. The
solvent was evaporated to provide a homogeneous
solid blend. The ýask was connected to a vacuum
line and cooled to liquid nitrogen temperature before
being ülled with tritium gas at 100 mbar pressure.
The neck of the ýask was connected to the 3Hhandling device located in a special glove box. The
bottom of the ýask was immersed in a thermostatted
silicone-oil bath and allowed to stand at 130¡C for
1 h. Eventually, the ýask was cooled to room temperature and the unreacted tritium was turned to
uranium tritide (UT ) using a dynamic vacuum with
nitrogen trapping. The ýask was then disconnected
and the reaction mixture dissolved by addition of
5 cm3 THF. The catalyst was removed by centrifugation and ültration using a 0.45 lm ülter. The solid
obtained after evaporation of the solvent was puriüed
by sublimation at 57¡C and was collected on the cool
wall of the tube.
Molecular weight determination
Molecular weights were determined by size exclusion
chromatography (SEC) using a Waters equipment
ütted with a 30 cm long 104 Ó Ultrastyragel column,
the mobile phase being THF delivered at a ýow rate
of 1 cm3 min~1, and a Waters 410 diþerential refractometer as detector with a ýow scintillation analyser
on-line (Flo-oneTM Beta Packard). Data were
expressed according to polystyrene (PS) standards.
The three selected polymers were synthesized
according to the same protocol.
12.5 mg of tritiated DL-lactide and 237.5 mg of
normal DL-lactide were blended (dilution 1/20) in a
1 cm3 round-bottomed ýask. A very small amount of
the selected initiator was then introduced (about
20 lg of Zn lactate or 20 mm3 of a 1 g dm~3 solution
of stannous octoate in THF). The small amount of
solvent was evaporated. The reaction mixture was
then melted at 140¡C and carefully degassed by the
vacuum/argon cycle method. Finally, the ýask was
sealed under vacuum by glass melting.
In the case of PLA100, we started from 12.5 mg of
tritiated L-lactide mixed with 237.5 mg of normal Llactide. Degassing was achieved at 120¡C.
217.5 mg of normal L-lactide and 20 mg of normal
DL-lactide were added to 12.5 mg of tritiated Llactide. Degassing was achieved at 120¡C.
Radiolysis assessment
The tritiated polymers were stored as a powder in a
refrigerator at 4¡C for 200 days. The radioactivity
was assessed occasionally during this period of time.
Typically, 2.5 mg of the radioactive polymer were
dissolved in 2 cm3 THF. Two aliquots of 10 mm3
were taken for radioactivity assessment using a
Packard Tri-Carb 1900 CA b-counter and BetamaxTM (ICN) as liquid scintillation cocktail. The
THF solvent of the rest of the initial solution was
distilled and collected using a liquid nitrogen trap.
Two 10 mm3 aliquots of the collected THF were
used to detect the presence of any radioactive volatiles using b-particle counting.
Hydrolytic degradation
Typically, a radioactive polymer was processed into
circular tablets (2 mm thick and 7 mm in diameter)
by compression moulding at 80¡C and 50 bar. In
parallel, 20 mm diameter thin ülms were made by
gently evaporating a concentrated PLA solution in
acetone from a Petri dish. After weighing, plate and
ülm specimens were introduced into identical ýasks
containing 20 cm3 of a phosphate buþer solution (pH
7.4, 0.13 M). The various ýasks were allowed to
stand at 37¡C in the same thermostatted oven. The
release of soluble radioactive species in the liquid
phase was monitored by two diþerent techniques,
namely liquid scintillation counting and capillary
zone electrophoresis (CZE) using a P/ACE 5000
Beckman instrument equipped with a UV detector
set at 200 nm and with fused silica capillaries (50 lm
diameter, 57 cm long). Only 40 mm3 of the liquid
phase was collected and split in two parts : 10 mm3
for radioactivity counting and 30 mm3 for CZE
analysis. For both analytical techniques, the data
were expressed in terms of lactyl repeating units for
the sake of comparison.
Polym Int 48 :283–287 (1999)
3H-labelled PLA polymers
Table 1. 1H ] 3H is otopic
exchange on L- and DL-lactides in
the pres ence of 5% Pd/CaCO
and tritium 100 mbar at 130¡C for
Specific activity
(Gbq mmol É1; Ci mmol É1)
Recovered lactide
1H ] 3H exchange a
150 (4.05)
111 (3)
a (Tritium fixed to lactide/number of tertiary carbon atom) ] 100
Tritiated lactide (DL or L) was synthesized according
to the best conditions obtained from a detailed investigation performed with deuterium.10 The speciüc
activity and the percentage of recovered lactide are
given in Table 1, together with the exchange yield.
For both compounds, the radioactivity was in the
range of 111–148 GBq mmol~1 (3–4 Ci mmol~1), a
value which is very high with respect to the levels of
radioactivity generally used in biology. The yields in
recovered lactide were close to 50%, a value which
was considered satisfactory. The substitution yield
was estimated from the lactide speciüc activity to be
in the range 5–7%, assuming that the exchange primarily involved the slightly acidic proton of the tertiary carbon atom present in the lactyl units, as
previously shown by NMR for deuteriated analogues.10 In the case of deuterium, it was possible to
increase the exchange yield up to 45% by using
higher pressures. However, corresponding high pressures could not be reached in the case of tritium, primarily
Nonetheless, the speciüc activity obtained was considered to be high enough to allow the synthesis of
the high speciüc activity PLA polymers required to
monitor the fate of the degradation by-products in
complex living systems, such as an animal body or an
outdoor medium. It is of value to note that the
obtained radioactivity was high enough to allow
further dilution of the tritiated lactides with normal
cold lactides up to any lower speciüc activity
required for a particular investigation.
The three selected monomer feeds were polymerized comparatively using stannous octoate and
Zn lactate whose mechanisms of action have been
comparatively investigated recently.11 The recovered
PLA50, PLA96 and PLA100 polymers had speciüc
activities in the range 29.6–44.4 MBq mg~1 (0.8–
1.2 mCi mg~1). The PS-related weight average
molecular weights M1 were in the range of 25 000–
Polym Int 48 :283–287 (1999)
Control of labelling stability
One of the main problems related to the use of radioactive compounds as tracers is radiolysis. Indeed,
radiolysis is generally a source of by-products which
can be later confused with the tracer itself, especially
when they become soluble in the surrounding ýuids
and can leach out from the polymer device. Another
problem is the possible presence of soluble radioactive remnants coming from the synthesis stage.
A speciüc protocol was selected to secure the
removal of tritium gas and other remnant lactides
from the substitution. The reaction media were ürst
submitted to dynamic vacuum to remove the excess
tritium gas. The solid residues were then dissolved
in THF in order to facilitate the release of the
tritium gas still adsorbed on the solid catalyst.
Lastly, the crude lactides were puriüed by sublimation, during which the solid radioactive residual
lactides were converted to a gas phase and turned
back into the solid state by condensation on a cold
wall. The speciüc activity of each lactide was quantiüed after the last puriücation step only. The preservation of a high level of radioactivity after all these
treatments was regarded as the ürst evidence in
favour of the stability of the labelling provided by
the proposed method. This conclusion was further
supported by the fact that the speciüc activity of the
polymers derived from the radiolabelled lactides was
preserved after polymerization and polymer puriücation.
For the sake of supporting this last statement, a
further investigation was performed on the PLA50
specimens. The level of radioactivity of the PLA50
Specific activity
(Gbq mmol É1;
Ci mmol É1)
(initiators )
(Zn lactate)
(Zn lactate)
(Zn lactate)
Table 2. Characteris tics of the
radiolabelled PLAs
60 000, M1 /M1 being lower than 2.5 for a conversion
w n
reaching about 75% (Table 2). These values of
molecular weight and molecular weight distribution
were considered to be convenient for the investigation of the radiolysis of the polymers and of their
sensitivity to hydrolytic chain cleavage.
M1 /M1
w n
Specific activity
(Mbq mmol É1 ;
mCi mmol É1)
41 000
57 000
48 000
25 000
I Dos Santos, J-L Morgat, M Vert
polymer stored at 4¡C was checked over a period of
200 days. During this period, polymer specimens
were taken occasionally and dissolved in THF. The
THF was recovered by distillation and its speciüc
activity was measured and considered as reýecting
the presence of radioactive volatiles such as tritiated
water. After 200 days, the cumulative distilled THF
radioactivity was only 0.45% of the total initial
radioactivity (Fig 1). It is of value to note that such a
low loss was impossible to detect by direct measurement of the speciüc activity of the aged polymer.
The monitoring of the polymer ageing by SEC using
two complementary detections, namely a diþerential
refractometer and a radioactivity detector, did not
show any signiücant variation in polymer molecular
weights and molecular weight distribution during the
200 day period.
Reliability of radiolabelled PLA
It has previously been shown that hydrolytic degradation of PLA polymers and stereocopolymers
depends on many related factors, including size and
shape. In particular, it was shown that thin devices
degrade much more slowly than thick ones, a paradoxical phenomenon which was well accounted for
by the so-called heterogeneous degradation mechanism.12 In order to take into account this possible
source of discrepancies, the degradation of radioactive compounds was studied for two types of devices,
namely 2 mm thick tablets and thin (about 0.3 mm)
ülms. The specimens were immersed in an isoosmolar pH 7.4 phosphate buþer solution at 37¡C,
and the radioactivity of the liquid medium was
assessed by liquid scintillation counting and by CZE
(Fig 2).
Liquid scintillation counting detected the release
of degradation by-products much earlier than CZE
and thus appeared to be a much more sensitive
method, as expected. However, CZE allowed the
identiücation of the released species when the
amount of released radioactive materials became high
enough. Accordingly, lactic acid, lactyl-lactic dimer,
and lactyl-lactyl-lactic trimer were detected, the
latter appearing in negligible amount.13 Using lactic
acid and lactyl-lactic acid standard solutions of
known concentrations, it was also possible to quantify the released species present in the phosphate
Figure 1. Cumulative los s of radioactivity vers us time in relation
to the initial radioactivity of the polymer.
Figure 2. Fractions of lactic acid releas ed during the degradation
of PLA tablets ÈÈ and films – – – in PBS (pH 7.4, 0.13 M) at 37¡C
as evaluated by b-counting (=) and by CZE (|) and expres s ed in
terms of percentage of the total lactic acid initially pres ent in the
s ample.
buþer solution by CZE and to compare the results
with radioactivity data (Fig 2). Similar degradation
proüles were provided by these two diþerent
methods. Furthermore, the comparison between the
proüles given by the tablet and the ülm-type specimens obtained from the same radioactive PLA50
sample appeared very diþerent, thus conürming the
previously reported eþect of size and shape on the
hydrolytic degradation characteristics of PLA polymers.12
Comparison was also made with data obtained previously for tablets and ülms made from a nonradioactive PLA sample with molecular weight
characteristics (M1 \ 37 000, M1 M1 \ 1.5) close to
w n
those of the radioactive PLA (M1 \ 41 000,
M1 /M1 \ 2.4). Degradation proüles appeared
w n
similar for both the radioactive and the nonradioactive specimens of the same type. Tablets
showed rapid release of low molecular weight species
after a long lag time. In contrast, more progressive
and long lasting release was observed in the case of
the ülms. The release of soluble degradation byproducts appeared earlier in the case of the radiolabelled specimens than in the case of the
non-radioactive ones (Fig 3). This release was
assigned to the greater polydispersity of the radioactive polymers, rather than to generation of oligomers
due to radiolysis. Indeed, it is known that the greater
the polydispersity, the faster the degradation.14 A
Figure 3. Fractions of lactic acid releas ed during the degradation
of radioactive PLA ÈÈ and non-radioactive PLA – – – in PBS (pH
7.4, 0.13 M) at 37¡C for tablets (=) and films (>) as meas ured by
CZE and expres s ed in terms of percentage of the total lactic acid
initially pres ent in the s ample.
Polym Int 48 :283–287 (1999)
3H-labelled PLA polymers
comparison between radioactive PLA50s of diþerent
molecular weight polydispersity should conürm this
interpretation in the future.
Stannous octoate and Zn lactate, the two initiator
systems used to polymerize the radioactive lactides,
led to similar polymers with similar stability and
hydrolytic degradation characteristics.15 This
absence of diþerence between the Sn- and the Zninitiated radioactive PLA50 contrasted with the previous ündings which showed that stannous octoate
leads to more hydrophobic and thus to more
hydrolysis-resistant PLA50 polymers than Zn metal
and Zn lactate.16 The greater hydrophobicity and the
longer lifetime of the polymers prepared with Sn
octoate were related to the esteriücation of some of
the polymer chain-ends by octanoic acid and also to
the presence of hydrophobic tin residues. Despite the
lack of accurate information, it is likely that the
absence of diþerence regarding the initiator eþects in
the case of radioactive compounds is somewhat
related to the very low monomer-to-initiator ratio
used to polymerize the radioactive lactides (M/
I \ 30 000).
An efficient method has been proposed for making
tritium radiolabelled lactides with very high radioactivity. The method is based on a catalysed isotope
exchange according to a protocol deüned in the use
of deuterium as a non-radioactive model labelling
compound. Both DL-lactide and L-lactide were
radiolabelled with speciüc activities in the 111–
148 GBq mmol~1 (3–4 Ci mmol~1) range. From the
tritiated L- and DL-lactides, radioactive PLA50,
PLA96 and PLA100 were obtained by ring opening
polymerization. The ürst studies on the stability of
these polymers show that the initial radioactivity was
retained over a period of 200 days on storage in the
solid state at 4¡C, only a negligible 0.45% of the
initial radioactivity being released according to liquid
scintillation counting. The monitoring of the appearance of soluble degradation by-products from tritiated PLA50 allowed to degrade hydrolytically in a
Polym Int 48 :283–287 (1999)
phosphate buþered saline (PBS) solution at 37¡C
conürmed the size dependence of the heterogeneous
hydrolytic degradation previously observed for nonradioactive PLA50 polymers. In the absence of artifactual release of radioactive compounds being
shown in PBS and in ethanol, tritiation of PLA polymers appears to be powerful means to investigate the
fate of PLA polymers with respect to the potential
applications of these polymers in surgery, in pharmacology, and probably in the environment, in the near
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amphiphilic, molecular, cryo, microdomains, cationic, medium, tem, roto, fluorescence, copolymers, aqueous, characterization, complementary, techniques
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