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doi:10.1016/j.bi

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Biomaterials 27 (2006) 4963–4974

www.elsevier.com/locate/biomaterials

The brain tissue response to biodegradablepoly(methylidene malonate 2.1.2)-based microspheres

in the rat

Elvire Fourniera,�,1, Catherine Passirania, Nathalie Colinb, Serge Sagodirab, Philippe Meneic,Jean-Pierre Benoita, Claudia N. Montero-Meneia

aInserm U646 (Ingenierie de la vectorisation particulaire), 10 rue Andre Bocquel, 49 100 Angers, FrancebHalisol, 46 rue Boissiere, 75 116 Paris, France

cDepartement de Neurochirurgie, CHU Angers, 4 rue Larrey, 49 100 Angers, France

Received 4 January 2006; accepted 28 April 2006

Abstract

The aim of this study was to follow the in vivo biodegradation as well as to appreciate the brain tissue response to poly(methylidene

malonate 2.1.2) (PMM 2.1.2)-based microspheres implanted into the rat brain. Ninety-three adult Sprague–Dawley female rats were

engaged in the study in which 54 underwent stereotactic implantation of blank g-sterilized PMM 2.1.2-based microspheres, prepared by

an emulsion-extraction method. Twelve rats were implanted with the same 5-fluorouracil (5-FU)-loaded microspheres. Seventeen

controls received the suspension medium alone (carboxymethylcellulose aqueous solution). The animals were sacrificed on post-operative

days 1, 2, 8 and months 1, 2, 3, 6, 9, 12, 15 and 18. The brains were dissected, frozen, cut in a freezing microtome, and the slides were

processed for immunohistological evaluation and scanning electron microscopy.

During the first few days, the moderate inflammatory response to blank or loaded PMM 2.1.2 microspheres was largely a consequence of

the mechanical trauma that occurs during surgery. The macrophagous-microglial reaction was similar to the one typically

found following any damage in the CNS. There were also no differences in GFAP reactivity between the implanted animals and the

controls. Blank microspheres began to degrade between 3 and 6 months, while 5-FU microspheres degraded between 8 days and 1 month.

The polymer degradation generated in both cases a pronounced inflammatory and immunological reaction, leading to an important cell

loss, a cerebral atrophy and to the death of several animals. PMM 2.1.2 was thus shown to be inadequate for intracerebral drug delivery.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Poly(methylidene malonate 2.1.2); Biocompatibility; 5-fluorouracil; Biodegradation; Microspheres; Rat brain

1. Introduction

The cerebral tissue is protected by the blood brain barrier(BBB), which limits the entrance of hydrophilic moleculeswithin the central nervous system (CNS). Hence, suchcompounds may require systemic administration at potentiallytoxic doses, in order to reach therapeutic levels in the brain.For improved delivery, different groups developed implan-

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.04.045

ing author.

esses: elvirefournier@hotmail.com,

serono.com (E. Fournier).

ess: Serono Pharmaceutical Research Institute, 14 chemin

Plan les Ouates, Geneva, Switzerland.

table polymeric drug carriers able to assure a site-specificsustained release of a drug, in various regions of the CNS[1–7]. Some systems were especially applied to the delivery ofanticancer drugs to the CNS in order to treat malignant braintumors, and underwent clinical trials [8,9]. Before testing thetherapeutic efficacy of a drug delivery device in clinical trials,researchers must first control the safety of the polymericmaterial and of its degradation products, in a convenient way[10]. Controlled release systems implanted in humans weretested before use in several biocompatibility studies performedin vivo in appropriate animal models [11–16].New poly(methylidene malonate 2.1.2) (PMM 2.1.2)-

based microspheres were developed in a previous study

ARTICLE IN PRESSE. Fournier et al. / Biomaterials 27 (2006) 4963–49744964

[17]. 5-fluorouracil (5-FU) was encapsulated as an idealcandidate for a site-specific sustained release, since it ishydrophilic and potentially toxic systemically. The effec-tiveness of these microspheres for an intratumoral che-motherapy approach against an established brain tumorwas demonstrated [18]. PMM 2.1.2 microspheres degradeslowly in vivo and could constitute an interesting long-termdelivery system for the treatment of neurodegenerativediseases. In this regard, the biocompatibility of thesemicrospheres with brain tissue should be explored.Biocompatibility data concerning PMM 2.1.2 nano ormicroparticles and their degradation products are availablein the literature, but their intracerebral behaviour has notbeen studied. The studies consisted mainly of cytotoxicityassays performed in vitro on different cell types but not oncells of the CNS [19–21]. One study was carried out in vivoin order to evaluate the short-term toxicity of intravenouslyinjected nanoparticles [22]. Thus, for an intracerebralapplication, biocompatibility was assessed on inadequatecells in vitro, and outside the CNS in vivo. Moreover, thelong-term biocompatibility of PMM 2.1.2 has never beenexplored.

Therefore, the goal of this study was to follow thebiodegradation of PMM 2.1.2-based microspheres im-planted into the rat striatum, and to evaluate the short-and long-term brain tissue responses to the implantation ofthe microspheres and to their degradation products. Forthis purpose, Sprague–Dawley female rats were injectedstereotactically with blank and loaded microspheres. Theywere thereafter sacrificed at different time points between 1day and 18 months, and their brains were compared tocontrols at each time point. Various immunostainings werecarried out in order to study the inflammatory andimmunological reactions, and scanning electron micro-scopy (SEM) was performed to observe the degradation ofthe microspheres.

2. Material and methods

2.1. Microsphere preparation and characterization

The microspheres were prepared using an emulsion/extraction method

previously described [17]. Briefly, PMM 2.1.2 (Mw 32,277) was dissolved

in methylene chloride (Prolabo, Paris, France) and an emulsion was

obtained by pouring the organic phase into a polyvinylalcohol aqueous

phase (2% w/v) (Rhodoviols 4/125, Prolabo, Paris, France) under

mechanical stirring. A large volume of deionized water was then added to

the emulsion, allowing the formation of microspheres, which resulted from

the solvent extraction. The particles were collected by filtration under

nitrogen pressure, washed with deionized water, frozen in liquid nitrogen

and freeze-dried. Finally, the particles were sterilized by g-irradiation at

25 kGy (Ionisos, Dagneux, France) and their size was determined by using

a Coulters counter (Multisizer, Coultronics, Margency, France).

The 5-FU microspheres were prepared in the same way. Before

dissolving the polymer (Mw 20,638), a suspension of 5-FU crystals was

formed in methylene chloride. Drug loading was determined by dissolving

microspheres in dimethylsulfoxide and reading by spectrophotometry the

optical density at 266 nm. The in vitro 5-FU release profile was carried out

in PBS pH 7.4 at 37 1C.

2.2. Animals

Sprague–Dawley female rats weighing 250–300 g were obtained from

the animal house of the Angers University Hospital. The animals were

kept in standard animal facilities with two rats/cage, and given free access

to food and water. They were housed in a temperature and humidity

controlled room with 12 h on–off light cycles.

2.3. Anesthesia

Animals were anesthetized with an intraperitoneal injection of

0.75–1.5ml/kg of a solution containing 2/3 ketamine (100mg/ml)

(Clorketams, Vetoquinol, Paris France) and 1/3 xylazine (20mg/ml)

(Rompuns, Bayer, Paris, France).

2.4. Microsphere implantation

For stereotactic implantation, the microspheres were sieved and a

suspension was obtained in a sterile aqueous medium consisting of 1.25%

(w/v) carboxymethylcellulose (CMC) (Cooper Pharmaceutique, France),

1% (w/v) polysorbate 80 (Prolabo, Paris, France) and 4% (w/v) mannitol

(Cooper Pharmaceutique, France). The suspension was sonicated before

use. Surgery was performed under aseptic conditions. The animals were

immobilized in a stereotaxic head frame (Lab Standard Stereotaxic,

Stoelting, Chicago, IL, USA) with the incisor bar set at �3.0mm. The

head was shaved, a midline incision was made in the scalp and a burr hole

was drilled at the following coordinates according to the atlas of Paxinos

and Watson [23]: 0.2mm posterior from Bregma, 3.0mm lateral from the

sagittal suture, and 5.0mm below dura.

Ninety-three animals were involved in the study. Fifty-four rats

underwent implantation of blank microspheres (2.5mg/10ml), 12 rats

received 5-FU microspheres (2.5mg/10ml) (10mg/kg 5-FU) and 17

controls received 10 ml of suspension medium.

All the injections were performed at a flow rate of 1ml/min, the cannula

was left in place for 4 additional minutes to avoid the suspension to be

expelled from the brain and the syringe was withdrawn very slowly

(0.5mm/min).

2.5. Histological examination

To evaluate the biodegradation and the biocompatibility of the

microspheres in the CNS, the animals were sacrificed on post-operative

days 1, 2, 8 and months 1, 2, 3, 6, 9, 12, 15 and 18. Until the 3rd month, 4

rats implanted with blank microspheres and 2 controls were sacrificed at

each time point. From the 6th month till the 18th month, 6 rats implanted

with blank microspheres and 3 controls were sacrificed at each time-point.

Rats implanted with 5-FU microspheres were sacrificed on post-operative

days 1, 2, 8, 30, 45 and 60. Two rats/point were sacrificed.

The brains were removed, frozen in isopentane cooled by liquid

nitrogen (1min, �40 1C) and stored at �80 1C. Frontal sections (20mm)

were performed in a freezing microtome (Leica, CM3050S, Nussloch,

Germany) at �20 1C throughout the whole implantation site. The sections

were recovered on slides and allowed to dry. The slides were kept at least

24 h at �20 1C before being processed for immunoperoxidase staining.

The sections were allowed to defrost at room temperature and were

then fixed with paraformaldehyde 4% (Sigma, Steinheim, Germany) for

15min at 4 1C and washed in PBS pH 7.4

The antibodies used are summarized in Table 1, and the immunohis-

tochemical procedure was carried out as follows. After washing in

phosphate-buffered saline (PBS), saturation of the non-specific sites was

performed with normal goat serum (Sigma, Steinheim, Germany) diluted

at 1/10 in PBS–BSA 4% (bovine serum albumin) (Sigma, Steinheim,

Germany) for 30min. Thereafter, sections were incubated with the first

antibody diluted in PBS–BSA 4% for 24 h at 4 1C (the anti-NOS antibody

was diluted in PBS–BSA containing 0.1% Triton). The incubation

with the second antibody, i.e. an anti-mouse or anti-rabbit (Vector

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Table 1

Antibodies used in the immunohistological procedure

Antibody Dilution Stained cells

Rabbit anti-glial fibrillary acidic (GFAP) protein polyclonal antibody

(Dako, Glostrup, Denmark)

1/200 Astrocytes

Mouse anti-rat CD11b monoclonal antibody (clone MRC OX42)

(Serotec, Blackthorn, Bicester, Bucks, UK)

1/100 Microglial cells, macrophages, dendritic cells, granulocytes

Mouse anti-rat ab T-cell receptor monoclonal antibody (BD

Biosciences Pharmingen, Heidelberg, Germany)

1/50 T lymphocytes

Mouse anti-rat CD4 monoclonal antibody (BD Biosciences

Pharmingen, Heidelberg, Germany)

1/50 MHC class II-restricted T cells (including most T helper

cells)

Mouse anti-rat CD8b monoclonal antibody (BD Biosciences

Pharmingen, Heidelberg, Germany)

1/50 MHC class I-restricted T cells (including most T

suppressor/cytotoxic cells)

Rabbit anti-nitric oxide synthase (NOS) type II polyclonal antibody

(BD Biosciences Pharmingen, Heidelberg, Germany)

1/100 Activated macrophages

Mouse anti-rat RT1B monoclonal antibody (clone OX6) (BD

Biosciences Pharmingen, Heidelberg, Germany)

1/100 Rat MHC class II antigens (B-lymphocytes, dendritic cells,

macrophages, activated microglial cells)

E. Fournier et al. / Biomaterials 27 (2006) 4963–4974 4965

Laboratories, Burlingame, USA) biotinylated antibody diluted at 1/100

was carried out for 1 h. To reduce the endogenous peroxidase activity, an

incubation was performed in PBS–H2O2 (Sigma, Steinheim, Germany)

0.3% for 15min. The amplification step was carried out with the

avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame,

USA) diluted at 1/100 in PBS for 1 h. For the anti-GFAP antibody, the

amplification step was not necessary and the second antibody was

peroxidase conjugated (Sanofi Diagnostics Pasteur, Marnes-la-Coquette,

France). The enzymatic reaction was developed with a solution of PBS

containing 0.03% of diaminobenzidine (DAB) (Sigma, Steinheim,

Germany) and 0.03% of H2O2 for 6–8min.

Three rinses of 5min each were performed between all incubations.

Several slides were counterstained with haematoxylin at the end of the

procedure. Finally the slides were washed with deionized water and

mounted with gelatin jelly. Control sections without the primary antibody

and isotypic controls were performed to verify the labeling specificity.

2.6. Microscopy studies

Slides were examined with an Axioscope 2 optical microscope (Zeiss,

Jena, Germany). Several slides were also viewed by SEM (Jeol JSM

6301F, Tokyo, Japan). Those ones were not stained. They were just

defrosted and metallized before SEM observation.

3. Results

3.1. Microsphere preparation and characterization

Blank and loaded microspheres were perfectly spherical.Their drug loading was between 17% and 24% (w/w).Their in vitro release profile showed a first rapid release of60% 5-FU within 24 h (‘‘burst effect’’), and a followingslow progressive release of 5-FU, which was still ongoingafter 43 days.

3.2. Biodegradation

3.2.1. Blank microspheres

One day after implantation in the rat striatum, themicrospheres appeared as regular and smooth spheresgrouped in a cluster. At day 8, an identical image wasobserved (Fig. 1a and b), and after 3 months, microspheres

were still perfectly intact and in a cluster (Fig. 1c). Theirsurface was still totally smooth and no pores weredetectable (Fig. 1d).After 6 months, the microspheres began to degrade from

the periphery to the center of the implant site, as shown inFig. 1e. In the center, microspheres could still be observed(Fig. 1f), irregular in shape, smooth but cracked. Thosemicrospheres were surrounded by totally degraded parti-cles, which had taken a polymer gel appearance.After 9 months, only PMM 2.1.2 gel could be seen and

the microspheres had totally disappeared. The gel hadtaken the same volume in the hemisphere, compared to theinitial volume of the microspheres, which indicated that itwas not resorbed (Fig. 1g). With higher magnification,pieces of microspheres could be seen and appeared asgranular particles which seemed to ‘‘melt’’ (Fig. 1h).

3.2.2. 5-FU microspheres

5-FU microspheres degraded between day 8 and 30.They were intact at day 8, their surface was smooth andthey were grouped in a cluster like blank particles. Invarious microspheres, 5-FU crystals could be observed bymicroscopy with polarized light or with differentialinterferential contrast (DIC) (Fig. 2a). The microsphereswere totally degraded at day 30. The inoculum (Fig. 2b)was still visible and appeared as a polymer gel whichpersisted at least until day 60. With higher magnification,the polymer gel was composed of particles around 20 mm indiameter, whose surface was granular (Fig. 2c and d). Nocrystals were observed with optical microscopy (differentialinterferential contrast or polarized light).

3.3. Biocompatibility

None of the animals exhibited any overt behavioralchanges, obvious neurological deficits or weight loss,suggestive of systemic or localized toxicity from thesurgical procedure or from the microspheres. Among the

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Fig. 1. Scanning electron micrographs of blank microspheres 8 days (a, b), 3 months (c, d), 6 months (e, f) and 9 months (g, h) after implantation in the rat

striatum.

E. Fournier et al. / Biomaterials 27 (2006) 4963–49744966

rats planned to be sacrificed at 18 months, 2 animals diedprecociously at 13 and 15 months, respectively.

3.3.1. Blank microspheres

At 24 h, activated microglial cells were observed directlyin contact with microspheres and around them, but not inthe whole striatum. These cells are characterized by ahypertrophic cell body and thick shortened processes,compared to resting microglia, with a small body and longprocesses. Only a few lymphocytes (o5) expressing ab

T-cell receptor were observed, close to the particles, andwere CD4+. No CD8+ cells were observed.At day 2, reactive microglial cells and monocytes/

macrophages were in contact with the microspheres(Fig. 3a), and activated cells were farther away. Reactivemicroglia are intensely stained and characterized by arounded amoeboid shape with sometimes very shortprocesses. They are difficult to distinguish from mono-cytes/macrophages. Some activated astrocytes with ahypertrophic cell body and long processes were observed,

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Fig. 2. (a) Optical micrograph with differential interferential contrast (DIC) 8 days after implantation in the rat striatum; 5-FU crystal are visible in

various microspheres (white arrowheads); (b–d) scanning electron micrographs (SEM) of 5-FU microspheres one month after implantation in the rat

striatum; the PMM 2.1.2 gel is made of granular degrading particles (white arrows in Fig. 2c); a higher magnification shows the surface of one particle (2d).

E. Fournier et al. / Biomaterials 27 (2006) 4963–4974 4967

not directly in contact with the microspheres (Fig. 3b) butaround the macrophagous cells, as illustrated on 2 adjacentsections. A few CD4+ lymphocytes (o5) were still present.

At day 8, GFAP immunoreactivity was maximum andastrocytes formed a ring around the microspheres (Fig. 3d),directly in contact with them. Activated microglial cellswere still present, around the astrocyte ring (Fig. 3c), butno macrophagous cells could be seen. A few CD4+

lymphocytes (o5) were still present.At 1, 2 and 3 months, astrocytes only persisted as

cicatricial tracks. Activated microglial cells could stillappear at 1 month, and were perfectly quiescent at latertime points (Fig. 3e and f).

No lymphocytes could be observed. No iNOS+ cells orcells expressing MHC class II molecules were observed atthose time points.

Concerning the control animals, the reaction wascomparable to the implanted animals, with a macropha-gous/microglial reaction maximum at day 2, and anastrocytic one maximum at day 8. A few CD4+

lymphocytes (o5) were also observed at days 1, 2, and 8.The animals sacrificed after 6 months presented an

extended and pronounced inflammatory reaction in theimplantation site. Reactive microglial cells and macro-phagous cells were located around the inoculum, infiltrat-ing the polymer gel and the microspheres, and covering thewhole striatum (Fig. 4a–c). An important astrocyticreaction was also observed around the inoculum, coveringthe whole striatum, but not infiltrating the degradingpolymer and the microparticles (Fig. 4b–d). Many lym-phocytes expressing the ab T-cell receptor were detectedand were mainly CD8+ (Fig. 5a–c). CD8+ lymphocytes

were both located in blood vessels and in the tissuesurrounding the polymer gel (Fig. 5d). iNOS positive cellscould be clearly observed in contact with the microspheresand within the polymer gel, and corresponded to OX-42positive cells (Fig. 5e). MHC class II immunoreactivity wasvery strong around the implant site and was expressed byboth OX-42 cells and astrocytes (Fig. 5f).Irrespective of the antibodies tested, the control animals

at 6 months were perfectly similar to the controls at 3months (Fig. 4e and f).After 9, 12, 15 and 18 months, microspheres were totally

degraded. The tissue was very fragile, but immunostainingswere nevertheless performed. At those time points, aninflammatory and astrocytic reaction was still highlypronounced, and lymphocytes could also be observed.The brains of the animals killed after 9 months weremacroscopically normal. The brains of the animals killedafter 12 months were macroscopically normal, but tissuedestruction of the injected side was observed when cuttingthe brains. The brains of the animals killed after 15 (Fig. 6aand b) and 18 months (Fig. 6c and e) presented variousdegrees of tissue destruction. Macroscopically normalbrains with some tissue loss that was visible when cuttingthe brain or macroscopically atrophied brains with tissuedestruction could be observed. One animal at 18 monthshad lost half of one hemisphere (Fig. 6d). The controlswere normal.

3.3.2. 5-FU microspheres

From day 1 till day 8, the inflammatory and immuno-logical reactions were identical to the blank microspheresand to the controls. The microglial reaction was maximal

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Fig. 3. CD11b antigen visualized by OX-42 antibody (a, c, e) and astrocytes visualized by anti-GFAP antibody (b, d, f), 2 days (a, b), 8 days (c, d) and 3

months (e, f) after implantation of blank microspheres. Rounded cells observed at day 2 are reactive microglial cells (arrow); at day 8, cells with a

hypertrophic cell body and thick short processes are activated microglial cells (arrowhead); at 3rd month, microglial cells are quiescent, i.e. cells with a

small body and long processes; astrocytes with a hypertrophic body are activated at day 8, at a distance from the microspheres (arrow); they encircle the

implant site at day 8 (arrowhead), and are quiescent at 3 months.

E. Fournier et al. / Biomaterials 27 (2006) 4963–49744968

at day 2 with rounded reactive microglial cells andmonocytes directly in contact with the microspheres. Theastrocytic reaction was maximal at day 8 and consisted ofactivated astrocytes forming a ring around the implant site,in contact with the microspheres. After 30, 45 and 60 days,the image was comparable to the reaction obtained at 6months with blank microspheres (Fig. 7). Numerousmicroglial and macrophagous cells were observed aroundthe micropsheres and infiltrating the polymer gel. Activatedastrocytes were also numerous around the implant site, butdid not infiltrate the polymer gel.

4. Discussion

4.1. Biodegradation

Biodegradation of the microspheres was followed in vivoby SEC, and showed that blank microspheres started todegrade between 3 and 6 months. Indeed, they were

perfectly intact after 3 months, and were partially degradedafter 6 months. At this time point, in the center of theimplant site, individual microspheres could still be clearlydistinguished, with a smooth and cracked surface. At theperiphery of the implant site, no microspheres could beobserved. They appeared as a polymer gel composed ofsmall granular particles. At 9 months, only the polymer gelwas observed, which was still not resorbed at 18 months.5-FU microspheres degraded much more faster. They

were still intact at day 8, but appeared as a polymer gelafter only 1 month. The different degradation ratesobserved between blank and loaded microspheres weredue in part to the different polymer molecular weights usedin the formulation of the blank and loaded particles. Twodifferent molecular weights were used consequently to aprevious optimization work which showed that a lowermolecular weight could slow down the release of thedrug [17]. Thus, loaded microspheres were formulated witha lower PMM 2.1.2 molecular weight for in vivo

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Fig. 4. CD11b antigen visualized by OX-42 antibody (a, c) and astrocytes visualized by anti-GFAP antibody (b, d), 6 months after implantation of blank

microspheres. Reactive microglial cells infiltrate the polymer gel and the microspheres (arrow in c). Activated astrocytes do not infiltrate the polymer gel

(arrow in fig.d). Microglial cells (e) and astrocytes (f) are quiescent on the controls.

E. Fournier et al. / Biomaterials 27 (2006) 4963–4974 4969

experiments, and therefore degraded faster, as classicallyobserved in the literature. Moreover, 5-FU is a hydrophilicmolecule. Hence, water can easily dissolve 5-FU crystalslocated at the periphery of the microspheres, which leads tothe creation of pores allowing the penetration of water inthe particle core and the dissolution of deeper 5-FUcrystals. Water thus rapidly penetrates in the polymermatrix, and hydrolyzes the polymer.

4.2. Biocompatibility

During the first days post-implantation, both injectedanimals (blank or loaded microspheres) and controlsshowed a brain tissue reaction similar to the one observedfollowing a mechanical lesion to the central nervoussystem. The microglial/macrophagous reaction was max-imal at day 2, and the astrocytic reaction at day 8. FewCD4+ lymphocytes patrolling in the CNS could be

observed (o5). In the case of blank particles, theinflammatory reaction thereafter decreased until onemonth, and glial cells remained quiescent until micro-spheres began to degrade. In the case of 5-FU loadedparticles, glial cells followed the same evolution pattern atleast until day 8. These results are in accordance withprevious publications concerning various drug carriersimplanted in the striatum such as PLGA microspheres[15,24–26], poly(e-caprolactone), ethylcellulose or polystyr-ene microspheres [27], poly[bis(p-carboxyphenoxy)pro-pane] and sebacic acid implants [11–14,28] and2-hydroxyethyl methacrylate copolymer microcapsules[29,30]. For all those controlled release systems, biodegrad-able or not, the brain tissue reaction after implantationconsisted of a typical and mild foreign body reaction whichresolved between 1 and 2 months.In this study, when microspheres began to degrade, the

inflammatory reaction was reactivated. At 6 months, the

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Fig. 5. T-cell receptor (a, b), CD8b (c, d), iNOS (e) and RT1B (f) antigens visualized by anti-TCRab, anti-CD8b, anti-iNOS and OX-6 antibodies,

respectively, 6 months after implantation of blank microspheres. Most lymphocytes (arrows) stay around the polymer gel (a, b); CD8+ lymphocytes (d)

are observed in vessels (arrow) or extravasated (arrowhead). iNOS positive cells (arrow) are observed between the degrading microspheres (e). OX-6

positive cells are located around, in contact and between the degrading microspheres (f).

E. Fournier et al. / Biomaterials 27 (2006) 4963–49744970

blank microspheres were not fully degraded since severalmicrospheres could still be observed in the center of theimplant site. At the periphery, the degraded microspheresconsisted of small granular particles which seemed to fuseand/or melt and took the appearance of a gel. Microglialcells, astrocytes, CD8+ lymphocytes, iNOS and CMHclass II positive cells were numerous and surrounded theimplant site. Microglial cells and iNOS-positive cells wereeven infiltrating the polymer gel and the degrading micro-spheres in the center of the implant site. At 1 month, the5-FU microspheres were totally degraded and a similartissue reaction was observed. Control brain cells were allperfectly quiescent. Hence, polymer breakdown productsclearly induced a direct toxicological effect on thesurrounding tissue, since the tissue reaction was ‘‘normal’’as long as the microspheres stayed intact. Moreover, thedamage was irreversible since an important tissue destruc-tion was observed in all the animals sacrificed at later time

points, and could lead to a macroscopically visible cerebralatrophy, and/or to the death of the animals.PMM 2.1.2 structure is represented in Fig. 8. Two

degradation pathways were detailed in previous studies[19,20]. The first one consists of the hydrolysis of the estergroups in the side chains, generating soluble polymers,ethanol and, to a lesser extent, glycolic acid. Indeed, the esterhydrolysis leads to the formation of free carboxyl groups onthe polymer backbone which becomes more and morehydrophilic, until it is water soluble, as previously describedfor poly(alkyl cyanoacrylates) [31]. The second pathway isthe backbone degradation process occurring through areverse Knoevenagel’s reaction similar to that detailed forpoly(alkyl cyanoacrylates) by Leonard et al. [32]. Thosepolymers can degrade by hydrolytic scission of the polymerchain to form formaldehyde and an alkyl cyanoacetate. Thispathway was shown to be minor in the PMM 2.1.2degradation process, compared to ester hydrolysis [19].

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Fig. 6. Rat brains 15 months (a, b) and 18 months (c–e) after implantation of blank microspheres into the rat striatum, just after sacrifice (a, c, d) and

during the cutting (b–e) (St ¼ striatum; cc ¼ corpus callosum; CaPu ¼ caudate putamen).

E. Fournier et al. / Biomaterials 27 (2006) 4963–4974 4971

Some studies have highlighted the low in vitro [19–21]and in vivo [22] toxicity of PMM 2.1.2 nanoparticles,compared to poly(alkyl cyanoacrylates). Moreover, pre-degraded particles were shown to be 3–5 fold less toxicthan non degraded particles, and ethanol and glycolic acidwere not toxic for the cells at the concentrations tested,which were much higher than what could be expected fromthe total degradation of the nanoparticles.

In this study, a severe toxicity of the PMM 2.1.2 wasobserved, which appeared just after the polymer began todegrade. Based on the studies mentioned above, this resultwas not expected. Nevertheless, different points mightexplain our results. Firstly, the biocompatibility of abiomaterial consists on its ‘‘ability to perform with anappropriate host response in a specific application’’ [33].Hence, biocompatibility strongly depends on the adminis-tration site. PMM 2.1.2 nanoparticles previously tested invivo [22] were injected intravenously to mice and rats, andassessed for acute toxicity. Organs were examined only 48 hpost-injection. Thus, nanoparticles did not induce anyshort-term pathological reaction, but the long-term effects

were not monitored. Secondly, cells tested in vitro werefibroblasts or Caco-2 cells, which are less sensitive thanneurons and even glial cells. Indeed, for our application, amore convenient in vitro cytotoxicity testing schedulewould have used a primary neuronal culture also contain-ing astrocytes, oligodendrocytes and microglial cells, sincea material should be evaluated on the cells with whom itwill be in contact in vivo.The soluble polymers generated by the ester hydrolysis

might have been responsible for the direct toxicity on cells,since they are acidic entities which seemed to resorb veryslowly in the cerebral tissue. The polymer degradationcontinuously generated acidic polyanions slowly resorbingand constituting a permanent inflammatory stimulus.Thereafter, the inflammatory and immunological reactioncontributed to the cytotoxic damage caused to the cells.Indeed, several iNOS-positive cells were observed, indicat-ing an ultimate activation state of the macrophagous cells.iNOS is an inducible nitric oxide-synthase which is notexpressed by CNS cells under physiological conditions. It isinduced in non-neuronal cells during various types of CNS

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Fig. 7. CD11b (a), astrocytes (b) and T-cell receptor (c) antigens

visualized by OX-42, anti-GFAP and anti-TCRab antibodies, respectively,

1 month after implantation of 5-FU microspheres. Reactive microglial

cells (arrow) infiltrate the polymer gel (a); activated astrocytes and

lymphocytes (arrows) stay around the implant site (b, c).

CH2

R1

R2

n

O

O

O

O

CH3

R1 =

O

O

CH3

R2 =

Fig. 8. Poly(methylidene malonate 2.1.2) chemical structure.

E. Fournier et al. / Biomaterials 27 (2006) 4963–49744972

injuries or diseases. Cells expressing iNOS in this case aremacrophages, microglia and astrocytes [34]. This enzymeallows the synthesis of the nitric oxide free radical gaswhich contributes to the elimination of bacteria andpathogens. Excess amounts can damage host cells, causingneurotoxicity [35]. In this study, iNOS was not expressedjust after implantation, which indicated a mild foreignbody reaction. However, during polymer degradation, NOsynthesis was detected and might be responsible for cellulartoxicity. Moreover, the potential second degradation

pathway of the polymer may become more important inthe brain tissue, and thus lead to a larger production of thetoxic compound formaldehyde.While degrading, the polymer also induced a specific and

pronounced immunologic reaction with many lymphocytesand a strong expression of MHC class II molecules. Aspecific immune response is usually observed following theintroduction of a foreign antigen in the body. For T-cells tobe stimulated, the foreign agent must be presented to themon the surface of an appropriate cell (antigen presentingcell (APC)) with a MHC molecule [36]. Synthetic polymersdo not constitute peptidic or glycolipid antigens, and thuscannot be processed by APC and presented to T-cells orNK T-cells (natural killers). Hence, other mechanismsmight be involved in the reaction observed in this study. Afirst hypothesis consists of the modification of endogenousproteins by the polymer, via hydrophobic interactions.Once denatured, proteins are generally not recognized bythe immune system. They are endocytosed, presented byAPC to specific T-cells, and activated T-cell clones maythus possess specificity against self-proteins, which maylead to cell lysis and death [36,37]. This hypothesis is

ARTICLE IN PRESSE. Fournier et al. / Biomaterials 27 (2006) 4963–4974 4973

supported by the presence of CD8+ lymphocytes. Indeed,exogenous antigen are generally processed and presentedvia the MHC class II pathway, while endogenous antigensuse the MHC class I pathway, which activates the cytotoxicT-lymphocytes (CD8+) [38,39]. Thus, we can suppose thatthe polymer altered endogenous proteins, involving theMHC class I pathway and activating CD8+ T-cells.

A second hypothesis is that polymer breakdownproducts can bind to appropriate carrier molecules in thesurrounding tissue, and become antigenic. Besides, theslowly degrading polymer constitutes a persistent source ofreaction products, which may induce the development ofan immunological memory [40].

Concerning the tissue destruction observed at long timepoints, different mechanisms can be proposed: firstly,activated CD8+ lymphocytes may be specific of selfproteins, leading to cell lysis, as mentioned above.Secondly, if phagocytic cells fail to engulf the foreignmaterial, they can nevertheless get close enough to causedegranulation. Enzymes are thus released in the surround-ing tissue causing destruction and promoting the inflam-matory reaction. Finally, macrophagous cells mayphagocyte small particles of the degrading polymer. Ifphagolysosomes fail to destroy the particles, or if theparticles of polymer are toxic for the cells, macrophagescan die and release highly toxic products in the surround-ing tissue such as oxygenated radicals and powerfulenzymes [40].

5. Conclusion

Blank and loaded PMM 2.1.2-based microspheres wereimplanted in rat brain in order to evaluate their biode-gradation and their biocompatibility. Microspheres firstappeared as a promising system, since they exhibited a slowdegradation rate. Indeed, blank microspheres were stillperfectly intact at 3 months. However, when the polymerstarted to degrade, a strong inflammatory and immunolo-gical reaction developed, leading to an important cell loss,a cerebral atrophy and to the death of several animals.Polymer degradation products thus generated directtoxicological effect on the brain tissue, and an interestingspecific immunological response. Further experiments arerequired to determine the potential toxic compounds, andto elucidate the mechanisms involved in the immunologicalreaction.

Acknowledgments

The authors want to thank Dr. A. Clavreul (InsermU-646, Angers, France) for fruitful discussions, and Dr.R. Filmon and M. Lesourd (Service commun de micro-scopie electronique, Universite d’Angers, France) for SEManalysis.

Appendix A. Supplementary materials

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.biomaterials.2006.04.045.

References

[1] Benoit JP, Faisant N, Venier-Julienne MC, Menei P. Development of

microspheres for neurological disorders: from basics to clinical

applications. J Control Release 2000;65:285–96.

[2] Menei P, Benoit JP, Boisdron-Celle M, Fournier D, Mercier P, Guy

G. Drug targeting into the central nervous system by stereotactic

implantation of biodegradable microspheres. Neurosurgery 1994;34:

1058–64.

[3] Brem H, Gabikian P. Biodegradable polymer implants to treat brain

tumors. J Control Release 2001;74:63–7.

[4] Gutman RL, Peacock G, Lu DR. Targeted drug delivery for brain

cancer treatment. J Control Release 2000;65:31–41.

[5] Gentile FT, Doherty EJ, Rein DH, Shoichet MS, Winn SR. Polymer

science for macroencapsulation of cells for central nervous system

transplantation. Reactive Polym 1995;25:207–27.

[6] Fung LK, Saltzman WM. Polymeric implants for cancer chemother-

apy. Adv Drug Deliv Rev 1997;26:209–30.

[7] Menei P, Montero-Menei C, Venier-Julienne MC, Benoit JP. Drug

delivery into the brain using poly(lactide-co-glycolide) microspheres.

Expert Opin Drug Deliv 2005;2:363–76.

[8] Brem H, Piantadosi S, Burger PC, Walker M, Selker R, Vick NA,

et al. Placebo-controlled trial of safety and efficacy of intraoperative

controlled delivery by biodegradable polymers of chemotherapy for

recurrent gliomas. Lancet 1995;345:1008–12.

[9] Menei P, Venier MC, Gamelin E, Saint-Andre JP, Hayek G, Jadaud

E, et al. Local and sustained delivery of 5-fluorouracil from

biodegradable microspheres for the radiosensitization of glioblasto-

ma: a pilot study. Cancer 1999;86:325–30.

[10] Fournier E, Passirani C, Montero-Menei C, Benoit JP. Biocompat-

ibility of implantable synthetic polymeric drug carriers: focus on

brain biocompatibility. Biomaterials 2003;24:3311–31.

[11] Brem H, Kader A, Epstein JI, Tamargo RJ, Domb A, Langer R,

et al. Biocompatibility of a biodegradable, controlled-release polymer

in the rabbit brain. Select Cancer Therap 1989;5:55–65.

[12] Brem H, Domb A, Lenartz D, Dureza C, Olivi A, Epstein JI. Brain

biocompatibility of a biodegradable controlled release polymer

consisting of anhydride copolymer of fatty acid dimer and sebacic

acid. J Control Release 1992;19:325–9.

[13] Tamargo RJ, Epstein JI, Reinhard CS, Chasin M, Brem H. Brain

biocompatibility of a biodegradable, controlled-release polymer in

rats. J Biomed Mater Res 1989;23:253–66.

[14] Olivi A, Kim KD, Benedetti L, Epstein JI, Pinn ML, Callegaro L,

et al. Ocular and brain biocompatibility of polymeric delivery

materials prepared from ester derivatives of hyaluronic acid. Clin

Mater 1993;13:163–8.

[15] Menei P, Daniel V, Montero-Menei C, Brouillard M, Pouplard-

Barthelaix A, Benoit JP. Biodegradation and brain tissue reaction to

poly(D,L-lactide-co-glycolide) microspheres. Biomaterials 1993;14:

470–8.

[16] Veziers J, Lesourd M, Jollivet C, Montero-Menei C, Benoit JP,

Menei P. Analysis of brain biocompatibility of drug-releasing

biodegradable microspheres by scanning and transmission electron

microscopy. J Neurosurg 2001;95:489–94.

[17] Fournier E, Passirani C, Colin N, Breton P, Sagodira S, Benoit JP.

Development of novel 5-FU-loaded poly(methylidene malonate

2.1.2)-based microspheres for the treatment of brain cancers. Eur

J Pharm Biopharm 2004;57:189–97.

ARTICLE IN PRESSE. Fournier et al. / Biomaterials 27 (2006) 4963–49744974

[18] Fournier E, Passirani C, Montero-Menei C, Colin N, Breton P,

Sagodira S, et al. Therapeutic effectiveness of novel 5-FU-loaded

poly(methylidene malonate 2.1.2)-based microspheres on F98 glioma-

bearing rats. Cancer 2003;97:2822–9.

[19] Lescure F, Seguin C, Breton P, Bourrinet P, Roy D, Couvreur P.

Preparation and characterization of novel poly(methylidene malonate

2.1.2.)-made nanoparticles. Pharm Res 1994;11:1270–7.

[20] Breton P, Roy D, Marchal-Heussler L, Seguin C, Couvreur P,

Lescure F. New poly(methylidene malonate 2.1.2) nanoparticles:

recent developments. In: Gregoriadis G, editor. Targeting of drugs.

New York, 1994. p. 161–172.

[21] Le Visage C, Couvreur P, Mysiakine E, Breton P, Bru N, Fattal E. In

vitro and in vivo evaluation of poly(methylidene malonate 2.1.2)

microparticles behavior for oral administration. J Drug Target

2001;9:141–53.

[22] Breton P, Seguin C, Roy D, Couvreur P, Lescure F. In vivo

evaluation of poly(methylidene malonate 2.1.2) nanoparticle toxicity.

In: Proceedings of the controlled release society 1994. p. 608–9.

[23] Paxinos G, Watson C. The rat brain in stereotaxic coordinates.

New York: Academic Press; 1986.

[24] Chen W, He J, Olson JJ, Lu DR. Carboplatin-loaded PLGA

microspheres for intracerebral implantation: In vivo characterization.

Drug Deliv 1997;4:301–11.

[25] Chen W, He J, Olson JJ, Lu DR. Direct intracerebral delivery of

carboplatin from PLGA microspheres against experimental malig-

nant glioma in rats. Drug Deliv 1998;5:101–10.

[26] Emerich DF, Tracy MA, Ward KL, Figueiredo M, Qian R, Henschel

C, et al. Biocompatibility of poly (DL-lactide-co-glycolide) micro-

spheres implanted into the brain. Cell Transp 1999;8:47–58.

[27] Menei P, Croue A, Daniel V, Pouplard-Barthelaix A, Benoit JP. Fate

and biocompatibility of three types of microspheres implanted into

the brain. J Biomed Mater Res 1994;28:1079–85.

[28] Brem H, Tamargo RJ, Olivi A, Pinn M, Weingart JD, Wharam M,

et al. Biodegradable polymers for controlled delivery of chemother-

apy with and without radiation therapy in the monkey brain.

J Neurosurg 1994;80:283–90.

[29] Campioni EG, Nobrega JN, Sefton MV. HEMA/MMMA micro-

capsule implants in hemiparkinsonian rat brain: biocompatibility

assessment using [3H]PK11195 as a marker for gliosis. Biomaterials

1998;19:829–37.

[30] Mokry J, Karbanova J, Lukas J, Paleckova V, Dvorankova B.

Biocompatibility of HEMA copolymers designed for treatment of

CNS diseases with polymer-encapsulated cells. Biotechnol Progr

2000;16:897–904.

[31] Lenaerts V, Couvreur P, Christiaens-Leyh D, Joiris E, Roland M,

Rollman B, et al. Degradation of poly(isobutyl cyanoacrylate)

nanoparticles. Biomaterials 1984;5:65–8.

[32] Leonard F, Kulkarni RK, Brandes G, Nelson J, Cameron JJ.

Synthesis and degradation of poly(alkyl alpha-cyanoacrylates).

J Appl Polym Sci 1966;10:259–72.

[33] Williams DF. Definitions in biomaterials. In: Williams DF, editor.

Progress in biomedical engineering. Amsterdam, 1987.

[34] Garcion E, Sindji L, Montero-Menei C, Andre C, Brachet P, Darcy

F. Expression of inducible nitric oxide synthase during rat brain

inflammation: regulation by 1,25-dihydroxyvitamin D3. Glia

1998;22:282–94.

[35] Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic

messenger. Ann Intern Med 1994;120:227–37.

[36] Remes A, Williams DF. Immune response in biocompatibility.

Biomaterials 1992;13:731–43.

[37] Rihova B. Biocompatibility of biomaterials: Hemocompatibility,

immunocompatibility and biocompatibility of solid polymeric mate-

rials and soluble targetable polymeric carriers. Adv Drug Deliv Rev

1996;21:157–76.

[38] Kuby J. Antigen processing and presentation. In: Freeman

WH and Company, editors. Immunology. New York, 1994.

p. 239–50.

[39] Newman KD, Elamanchili P, Kwon GS, Samuel J. Uptake of

poly(D,L-lactic-co-glycolic acid) microspheres by antigen-presenting

cells in vivo. J Biomed Mater Res 2002;60:480–6.

[40] Williams DF. Tissue-biomaterial interactions. J Mater Sci 1987;22:

3421–45.