Degradable polymers in a living environment: where do you end up? †

Polymer International 51:840-844 (2002) Oct02

Michel Vert *, Isabelle Dos Santos, Stéphanie Ponsart, Nathalie Alauzet, Jean-Louis Morgat, Jean Coudane, Henri Garreau Research Centre for Artificial Biopolymers, UMR CNRS 5473, University Montpellier 1, Faculty of Pharmacy, 15 Ave Charles Flahault, 34093 Montpellier Cedex 05, France

email: Michel Vert (vertm@pharma.univ-montp1.fr)

*Correspondence to Michel Vert, Research Centre for Artificial Biopolymers, UMR CNRS 5473, University Montpellier 1, Faculty of Pharmacy, 15 Ave Charles Flahault, 34093 Montpellier Cedex 05, France

† Plenary lecture: Paper presented at the Polymers in the Third Millennium Conference, 2-6 September 2001, Montpellier, France.

Research Centre for Artificial Biopolymers, UMR CNRS 5473, University Montpellier 1, Faculty of Pharmacy, 15 Ave Charles Flahault, 34093 Montpellier Cedex 05, France

Abstract

The resistance of synthetic polymers to the action of living systems is becoming more and more problematic in certain domains in which they are used for a limited period of time before becoming waste. It is exemplified in surgery, pharmacology, agriculture and in the environment too. In these domains, time-resistant polymeric wastes are less and less acceptable. From this viewpoint, sutures, bone fracture fixation devices, mulch films and packagings are comparable. Basically they should be eliminated after use. Post-use biorecycling is regarded as a possible solution to some of the problems raised by the management of these polymeric wastes, regardless of the domain of application. This contribution aims to present simple and versatile methods with a potential to investigate the fate, and especially the bioassimilation, of the degradation by-products of degradable or bio-degradable polymers in complex living media such as the human body, a compost or the outdoor environment. Two versatile methods are presented that have been developed to radio-label degradable and biodegradable artificial aliphatic polyesters by substituting some protons by tritium atoms. It is also shown that weighing a population of starved earthworms, allowed to be in contact with degradable or biodegradable polymer, is a worthwhile method to demonstrate that degradation by-products are bioassimilated. 

Keywords: degradable polymers; biodegradable polymers; bioassimilation; polyester radiolabelling; earthworms

http://www3.interscience.wiley.com/cgi-bin/abstract/99019806/ABSTRACT 

For the last 60 years, synthetic polymeric materials have grown progressively to form one of the more attractive domains in polymer science. During this period, they have invaded almost every human activity. This success is due primarily to their low cost, their reproducibility at high speed, and their durability related to a high resistance to physical ageing and biological attacks. However, the resistance of synthetic polymers to the action of living systems is becoming more and more problematic in several domains where they are used for a limited period of time before becoming waste. This is the case in surgery, in pharmacology, in agriculture and in the environment as well. In these domains, time-resistant polymeric wastes are less and less acceptable. From this viewpoint, sutures, bone fracture fixation devices, mulch films and packagings are comparable. Basically they should be eliminated or recycled after use in cases where they can be recovered. However, there is a number of situations where recovery is not easy or feasible, like in surgery for obvious reasons, or in the environment in the case of litter. Moreover, recycling by reprocessing or via chemistry after degradation to generate exploitable chemicals is still an unsolved issue.

Among the various possible routes to eliminate polymeric wastes, biodegradation and biorecycling via bioassimilation are regarded as attractive solutions, especially in surgery and pharmacology where biostable wastes have to be left in place or retrieved, or for environmental protection when incineration is not feasible, either because it is a source of unacceptable pollution or simply because it is not feasible as in surgery. During the last 30 years people have been looking for new polymeric materials which could be eliminated after use and, ideally, biorecycled. However, turning a synthetic polymer, which is not a pollutant, into a batch of degradation by-products may not be innocent in so far as biocompatibility is concerned. Literature is full of novel artificial polymeric compounds that are said to be degradable and/or biodegradable on the basis of their degradation behaviour under conditions modelling the complex living media in which they are supposed to be degraded.[1] However degradation and biodegradation phenomena do not imply that the degradation byproducts are harmless for the living media and are mineralized and/or bioassimilated in the end. Therefore, in designing so-called biodegradable polymers, one must take into account the fate of polymer residues and not only the mechanism through which the material breaks down.

From a general viewpoint, regardless of the type of applications, biodegradability is monitored through direct and indirect methods aimed at showing property changes. Visual examination by optical or by scanning electronic microscopy and measurements of molecular weight changes and of weight loss are the most commonly used strategies. Monitoring the fate of the degradation by-products is much more difficult when these products are processed in complex living media such as a human body, a compost or in the outdoor environment. Whenever degradation is to occur under outdoor environmental conditions, ie through microorganism activity, measuring the amounts of oxygen or CO₂ that are respectively consumed or produced during biodegradation by respirometry is the most commonly used strategy. However data are often perturbed by contributions due to the living biomass present in the overall system. In the case of degradation occurring in a volume-limited living system like a human or an animal body where high molecular weight compounds are normally entrapped between skin and mucosa, with an exit pathway through the kidneys the radiolabelling is the best technique to monitor the fate of a foreign polymer, according to protocols already applied in pharmacology. Basically the same strategy can be applied to polymers that degrade under outdoor conditions. The major problem is then related to having a radiolabelled polymer that corresponds to the normal material to be tested, noting that radioactive chemistry requires special radiosynthesis equipment. A closely related problem is found in pharmacology, in the case of testing the activity of an antitumoral drug via monitoring tumour growth or tumour regression for instance. By simply weighing the mouse, it is possible to show whether the drug is active or not. Starting from this statement, we came to the idea that a similar strategy could be applied to test the ability of an animal organism to degrade and/or biodegrade and then bioassimilate an artificial polymer, the basic concept being that a starved animal will gain weight if it is in the presence of a degradable polymer that is a nutrient or that generates nutrients, otherwise it will lose weight.

In this contribution, we wish to present and discuss the methods we have developed during recent years with the aim of having tools to monitor the fate of polymer degradation by-products and their bioassimilation in complex living media. In a first part, versatile methods to radiolabel aliphatic polyesters of the poly(lactic acid), PLA, and of the poly(e-caprolactone), PCL, families by tritiation will be discussed. In the second part the use of a starved population of earthworms to show the bioassimilation of degradation by-products issued from a degraded polymer will be presented, PLA being taken as example of degradable polymer.

RADIOLABELLING OF ALIPHATIC POLYESTERS BY TRITIATION

Among various methods that can be used to introduce hydrogen isotopes, ie deuterium and radioactive tritium , into an organic molecule, isotopic exchange in the solid state proposed by Zolotarev et al using the HSCIE method, [2,3] appeared very efficient in the case of amino acids and peptides. This method is based on the reaction of deuterium or tritium gas on a highly dispersed solid compound intimately mixed with a metal catalyst such as rhodium, palladium, platinum on an inorganic carrier such as barium sulfate, potassium carbonate, aluminum oxide or coal. As the polymer to be radiolabelled is supposed to be degraded in a complex living medium with a family large dilution, the initial specific radioactivity has to be initially as high as possible. The use of radioactive ¹⁴C atoms to label lactic acid-containing aliphatic polyesters was reported some time ago.[4] However, radioactivity level in that procedure was rather low and suitable for small animals only. Monitoring the fate of degradation products after dispersion in large complex media such as large animals, used to model the human body, or the outdoor environment, requires much higher radioactivity. For this reason we decided to set up a radiosynthesis laboratory approved for tritiation at high radioactivity (Fig 1). The Zolotarev method failed in the case of poly(DL-lactide) because of dramatic main chain degradation. However, it was successfully applied to DL-lactide as recently reported.[5]

For the sake of avoiding the security constraints related to the handling of highly radioactive chemicals, most of the experiments aimed at finding high reaction yields and structural characteristics were performed with deuterium gas, the non-radioactive isotope of hydrogen.

Figure 1. View of the special radiosynthesis laboratory and glove box set up to handle and carry chemical reactions with highly radioactive tritium gas.

Scheme 1. H —>  H  exchanges on lactide molecules.

In a first set of experiments, several palladium and platinum catalysts on different supports were tried, namely 10% Pd/charcoal, 5% Pd/CaCO₃, 10% Pd/A1₂0₃, 10% Pd/BaSO₄, 5% Pd/diatomaceous earth, Pt0₂, temperature and deuterium pressure being fixed at 90°C and 550mBar, respectively.[5] The exchange occurs without racemization. Conditions to obtain significant hydrogen-deuterium exchange and limited degradation were found by varying the catalyst and reaction temperature and pressure. 5% Pd/CaCO₃ was selected as the most convenient catalyst. Although reaction temperature and pressure influenced the exchange and degradation yields, the dispersion of the solid turned out to be the most critical factor.[5] This finding required the increase of the surface of the rather small round bottom flask used as reaction vessel in order to favour isotopic exchange. This was achieved by filling the flask with small glass beads. The method was also efficient in the case of hydrogen-tritium exchange but led to lower exchange yields.[5] Mass spectra and NMR analyses on nonradioactive deuteriated lactides taken as model compounds showed that substitution occurred with a 42% yield and involved the methyne proton and the reaction yielded a 40/38/22% blend of zero, mono- and disubstituted lactides, respectively (scheme 1).[5] Glycolide was tritiated using the same technique.[6] The method yielded a 33/40/21/5 % blend of zero, mono, di and tri substituted glycolides, respectively. From both the radioactive lactides and glycolide, radioactive PLA and polyglycolic acid and lactic acid-glycolic acid copolymers were prepared that are now available for experiments in complex media.[7] For instance, highly radioactive poly (DL-lactic acid) (specific radioactivity 4.181 x 10⁷ Bq mg g ¹) was used to monitor the time-dependence of the body distribution of poly(ethylene glycol)-grafted stealth nanoparticles made of poly (DL-lactic acid).[8]

Scheme 2. Aniomic activation of PCL using LOA (step 1) and substitution by Rx (step 2).

Substitution reactions via anionic activation by proton extraction at the a-position with respect to the carbonyl of an ester group by using a non-nucleophilic base has been known for long time in organic chemistry.[9] The method had not been extended to aliphatic polyesters until it was applied to PCL by our group,[10] probably because of the sensitivity of these macromolecules to lysis. From a general viewpoint, the substitution occurs in two steps via the equations in Scheme 2. Step 2 corresponds to the reaction of an electrophile with the carbanion sites created on the PCL chain during step 1. Despite some polymer chain cleavages due to side reactions, the polymers recovered were still high molecular weight compounds. The method was used to react tritiated water as the electrophile to substitute a proton with H and thus make PCL radioactive.[11] A radioactive PCL was obtained that had a specific radioactivity of 152 µCi g ¹. This polymer was used to investigate the fate of PCL degradation by-products when the polymer is allowed to stand in the presence of microorganisms in a culture medium inoculated by an activated sludge collected from the Cereirède sewage plant in the Montpellier area in France.[12] Degradation profiles of radioactive PCL in inoculated culture medium showed that the lag time was much smaller than the lag time reported from respirometry measurements thanks to the sensitivity of radioactivity detection. After 16 day incubation, capillary zone electrophoresis showed that no water-soluble oligomer was present in the ageing medium. About 80-90% of the radioactivity initially present in the solid PCL was recovered as tritiated water depending on the run. It was further shown that the remainder of radioactivity was incorporated into the biomass sticking to the vessel wall. These features show conclusively that after 72 days at 37°C PCL was 100 % biodegraded.

Figure 2. Jar used to allow earthworms to live in an artificial culture medium without carbon or with the degradable compound to be tested regarding bioassimilation.

The method was also applied to PLA polymers (Ponsart, Caudane, Morgat and Vert unpublished). However the side reactions caused a dramatic decrease of molecular weight and significantly limited the available degree of substitution

As stated in the introduction, a method was recently developed in our group to monitor the bioassimilation in animals present in outdoor media like compost or ground, instead of bacteria and/or fungi, the microorganisms being equipped with enzymes that are not present in animals. The method is based on the comparison between the variation in weight with time of a population of starved adult earthworms that lose weight regularly with time and a similar population initially deprived of nutrients, and then fed with the degradable or biodegradable polymer to be tested. The latter gains weight if the polymeric compounds or its degradation by-products are nutrients and are thus bioassimilated.[13] In a typical experiment, adult or subadult Eisinia andrei earthworms, generally used in lombricomposting, were placed in a jar (schematically represented in Fig 2) where they were allowed to fast on a mineral support (Levilite) for 62 days, ie the average time to observe 50% weight loss. It is worth noting that the starved worms survived beyond 140 days, the weight loss reaching 80% of the initial value (Fig 3). At 62 days, populations of 10 worms were taken and placed in a similar jar loaded with an artificial culture medium adapted to worm growing and named Biosynthesol. This medium combined Levilite and glass balls used as mineral support, ammonium nitrate as source of nitrogen, a small amount of glucose and a supply of mineral salt known as Winogradsky medium.[14] Figure 3 shows the response of such a population of starved worms when the medium stayed carbon-free and when a biodegradable and bioassimilable polymer like cellulose was introduced as a source of carbon. For the carbon-free system, the weight of the population continued to decrease whereas the population exhibited a clear weight gain for the period of time during which bioassimilation occurred and then the weight decreased again when the carbon source was exhausted. This method was applied to high molecular weight PLA96 and PLA50, and to oligoPLA96 and 50 having weight average molecular weights of 97 000, 165 000, 730 and 825 g mol^-1 respectively. None of the two high molecular weight compounds caused weight gains whereas the two oligomers were bioassimilated and allowed the population to regain its initial weight within 3-4 weeks before loosing weight again. This is a very simple method that can be applied to any polymer. The data were easily correlated to what was already known from abiotic in vitro degradation and from the degradation of PLAGA polymers of different characteristics in the presence of micro-organisms. 13 it has been suggested that high molecular weight PLA cannot be degraded by earthworms. It is likely that polymers have first to be degraded abiotically, then bioassimiated by microorganisms and that they are then assimilated by the worms, a mechanism that has still to be demonstrated conclusively.

Figure 3. Average weight changes with time of a population of earthworm in the absence of nutrient and in the presence of a biodegradable polymer, cellulose.

Figure 4. Autoradiograph of a worm allowed to live in a model culture medium for 12 days in the presence of a 14C-radiolabelled sample of oligoPLA50, obtained with Cyclone®' photoimager.

Monitoring bioassimilation in animals through radioactivity

As stated above, radioactivity measurements are basically a powerful method to demonstrate the fate of polymer degradation by-products. However care must be taken with the label location. For example, we have previously shown that PLA polymers can be labelled with tritium by the HCSIE or by anionic activation, both methods placing the tritium atom onto the tertiary carbon atom. Basically, such tritiated PLA can be used to show the bioassimilation of the polymer. However there is a risk of false negative result if one looks only to the radioactivity of the biomass. As soon as it penetrates a cell, lactic acid is turned to pyruvic acid and thus loses the tertiary hydrogen isotope present to form tritiated water. An alternative strategy then is to use ¹⁴C labelling despite the poorer b-emitting yield of this nucleus compared with tritium. Figure 4 shows the autoradiograph of a worm fed for 12 days with oligoPLA50 radiolabelled with ¹⁴C at all carbon positions. Under these conditions, radioactive CO₂ is formed and can be assessed to reveal the biochemical processing of the oligomers in the medium but not necessarily by the worms.[13]

CONCLUSION

It has been known for long in biochemistry that bioassimilation of chemicals can be monitored using radiolabelling and/or biomass formation. In the case of artificial degradable or biodegradable polymers, the literature is primarily based on indirect methods such as respirometry involving micro-organisms like bacteria and fungi. In this presentation, it has been shown that rather easy methods based on radiolabelling and assessment of biomass formation using earthworms are now available that allow people to investigate the fate of degradation by-products in the complex living systems where degradation occurs. Both the HCSIE and the anionic activation tritiation processes, as well as the earthworm-based assessment of biomass formation, are versatile methods in the sense that they can be applied to a large number of polymers.

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