Acta Biomaterialia 10 (2014) 1194–1205
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Poly(ethylene glycol) methacrylate hydrolyzable microspheres for transient vascular embolization Stéphanie Louguet a, Valentin Verret a,b, Laurent Bédouet a, Emeline Servais a, Florentina Pascale b, Michel Wassef c, Denis Labarre d,e, Alexandre Laurent c,f, Laurence Moine d,e,⇑ a
Occlugel S.A.S., 12 Rue Charles de Gaulle, 78350 Jouy en Josas, France Archimmed S.A.R.L., 12 Rue Charles de Gaulle, 78350 Jouy en Josas, France AP-HP hôpital Lariboisière, Department of Pathology, University of Paris 7 – Denis Diderot, Faculty of Medicine, 2 rue Ambroise Paré, 75010 Paris, France d Université Paris-Sud, Institut Galien Paris-Sud, LabEx LERMIT, Faculté de Pharmacie, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France e CNRS UMR 8612, Institut Galien Paris-Sud, LabEx LERMIT, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France f ‘‘Laboratoire Matières et Systèmes Complexes’’, CNRS 7057, University of Paris 7, Bâtiment Condorcet, 10 rue Alice Domon et Léonie Duquet, 75205 Paris Cedex 13, France b c
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Article history: Received 25 June 2013 Received in revised form 6 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Embolization Microsphere Degradable Hydrolyzable crosslinker PEG-based hydrogel
a b s t r a c t Poly(ethylene glycol) methacrylate (PEGMA) hydrolyzable microspheres intended for biomedical applications were readily prepared from poly(lactide-co-glycolide) (PLGA)–poly(ethylene glycol) (PEG)–PLGA crosslinker and PEGMA as a monomer using a suspension polymerization process. Additional co-monomers, methacrylic acid and 2-methylene-1,3-dioxepane (MDO), were incorporated into the initial formulation to improve the properties of the microspheres. All synthesized microspheres were spherical in shape, calibrated in the 300–500 lm range, swelled in phosphate-buffered saline (PBS) and easily injectable through a microcatheter. Hydrolytic degradation experiments performed in PBS at 37 °C showed that all of the formulations tested were totally degraded in less than 2 days. The resulting degradation products were a mixture of low-molecular-weight compounds (PEG, lactic and glycolic acids) and water-soluble polymethacrylate chains having molecular weights below the threshold for renal filtration of 50 kg mol1 for the microspheres containing MDO. Both the microspheres and the degradation products were determined to exhibit minimal cytotoxicity against L929 fibroblasts. Additionally, in vivo implantation in a subcutaneous rabbit model supported the in vitro results of a rapid degradation rate of microspheres and provided only a mild and transient inflammatory reaction comparable to that of the control group. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Embolization, i.e. occlusion of arteries feeding organs with injected biomaterials, is a technique used for the treatment of various pathologies such as tumors, arterio-venous shunts and hemorrhages. It is commonly achieved with particles, which can be either degradable or non-degradable. The degradable particles are preferred when a durable vessel occlusion is not required, for example in uterine fibroids that are very sensitive to ischemia, in bleedings where the blood leak can be stopped if the flow is blocked for a few hours, or in some tumors, specially malignant ones, when embolization has to be repeated in the same feeder arteries. Non-degradable particles have evolved considerably in the last few decades. The irregular poly(vinylalcohol) (PVA) particles of the
⇑ Corresponding author at: Université Paris-Sud, Institut Galien Paris-Sud, LabEx LERMIT, Faculté de Pharmacie, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France. E-mail address: [email protected] (L. Moine).
1970s, which were poorly calibrated, aggregated proximally in clusters and gave a chronic inflammatory response [1–3], have been replaced by smooth, biocompatible and calibrated microspheres (MS). They drastically changed the conditions of embolization, since the radiologist could adapt the size of the MS to the size of the vessels to allow precise targeting and accurate devascularization. These MS have since been chemically grafted, to be loaded ionically with cationic drugs, and have thereby become actual drug delivery systems for local chemotherapy in tumor embolization [4,5]. By comparison, the evolution of the degradable particles has been very slow. Porcine sponge particles (GSP) were used at the very beginning of embolization in the 1970s and are still commonly used today. The enzymatic degradation of GSP is highly variable over time, from 3 weeks to 4 months [6–10], and is accompanied by a chronic inflammatory response and a vessel remodelling process [6,9], so that functional recanalization cannot be assured [11,12].
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.11.028
S. Louguet et al. / Acta Biomaterialia 10 (2014) 1194–1205
It is only very recently that two types of degradable MS have become available for embolization. However, their severe limitations hamper their competitiveness with non-degradable MS. Starch MS (Embocept™, Pharmacept) have been proposed to enhance drug diffusion in tumoral tissues when coadministered with chemotherapeutic drugs [13–15]. However, their small diameter (500 lm). Collagen-coated poly(lactide-co-glycolide) (PLGA) MS (Occlusin™500, IMBiotechnologies) are also available, but in only one small size (150–212 lm). Further, their in vivo degradation requires several months and is accompanied by an inflammatory and fibrotic reaction [16]. We have therefore conceived a degradable microsphere that encompasses the useful properties of non-degradable MS. First, we aimed to make a degradable microsphere which temporary occludes the vessel between a few hours and a few days, which is sufficient to achieve an ischemia of tumors (in the case of fibroids), a vessel repair (in the case of bleeding) or the delivery of a drug to tumors (in the case of chemoembolization). Second, this biomaterial should, after its degradation, be eliminated quickly, before the onset of a chronic inflammatory response and vessel wall remodelling [l2]. Third, the MS must be calibrated in several size ranges, between 100 and 1000 lm. Fourth, the MS should be easily suspended in physiological solutions and contrast medium. Fifth, the material must be soft and elastic enough to be injected via microcatheters of diameters smaller than the MS, and to recover their size and shape upon exit [17]. Sixth, the MS must be loadable with drugs for transient chemoembolization procedures. To fulfill the above-listed requirements, we have successfully developed a tunable and degradable MS that combines poly(ethylene glycol) methacrylate (PEGMA) and a hydrolyzable crosslinker (PLGA–poly(ethylene glycol) (PEG)–PLGA). PEGMA was selected as the monomer because of its amphiphilic nature, which results from its water-soluble PEG side chain and its hydrophobic methacrylate group. The amphiphilic nature of PEGMA makes possible mixing it directly with the hydrolyzable crosslinker in hydrocarbon phase to perform the polymerization by direct oil in water suspension process for making microspheres. The hydrolyzable crosslinker was designed to achieve rapid degradation time. An anionic co-monomer, methacrylic acid (MA) was combined to the PEGMA monomer and the crosslinker to adjust the water uptake and to allow the ionic loading of drugs. A large part of the strategy was devoted to the biocompatibility of the material. First, the initial components were chosen to generate water-soluble macromolecules after hydrolysis in order to avoid any accumulation in the organism, which might lead to inflammatory reactions [18]. Second, additional ester linkages from a cyclic ketene acetal monomer, 2-methylene-1,3-dioxepane (MDO), were incorporated into the hydrogel backbone to shorten the size of the polymer degradation products (below 50 kg mol1), facilitating their renal elimination [19,20]. We report here the feasibility of preparing suitable degradable microspheres for embolization with the selected components (hydrolyzable crosslinker, PEGMA, MA and MDO). The impact of each component on the degradation time and on the size of the degradation products was evaluated. The hydrolytic degradation of the different formulations was characterized and the effect of degradation products on the biocompatibility was studied by two different cytotoxic tests. Subcutaneous implantations were then performed in rabbits to assess the in vivo degradation of MS and the local inflammatory reaction.
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2. Material and methods 2.1. Materials PEGMA (Mn = 300 g mol1), stannous octanoate, triethylamine, methacrylic anhydride, 88% hydrolyzed PVA, glycolide, 1-hexanethiol and polyethylene glycol (Mw = 600 g mol1) (PEG) were purchased from Aldrich (St Quentin Fallavier, France). 2,20 -Azobisisobutyronitrile (AIBN), used as a polymerization initiator, and trimethylsilyldiazomethane (2 M in hexane; TMS) were obtained from Acros Organic (Geel, Belgium). D,L-Lactide was obtained from Biovalley (Marne la Vallée, France). MDO was synthesized according to the procedure published in Ref. [21]. Doxorubicin hydrochloride (Adriblastin) was purchased from Pfizer (France). Embosphere (non-degradable gelatin microsphere; GMS) was purchased from Biosphere Medical (Roissy, France). DC-beadsTM microspheres were purchased from Biocompatibles (Farnham, UK). All reagents were used without any further purification. Analytical grade solvents were supplied by Carlo Erba (Val de Rueil, France). 2.2. Instrumentation 2.2.1. Nuclear magnetic resonance (NMR) Products were analyzed by 1H NMR spectroscopy using a Bruker DPX300 FT NMR spectrometer, with the solvent peak as a reference. 2.2.2. Fourier transform infrared spectroscopy (FTIR) FTIR was used to characterized the chemical structure of polymers and MS. Spectra were recorded on a Spectrum One Perkin Elmer infrared spectrometer equipped with a diamond attenuated total reflectance module in a range of 500–4000 cm1. One hundred interferograms were averaged per spectrum at a resolution of 2 cm1. 2.2.3. Size exclusion chromatography (SEC) The molecular weight and molecular weight distribution of polymers were measured by SEC at 30 °C on a system equipped with a guard column and two GMH HRm columns (Viscotek) with tetrahydrofuran (THF) as the eluent at 1 ml min1. A differential refractometer and a double SEC detector (model 270 Dual, Viscotek) with RALS and viscosimeter in series were used to analyze samples. The data obtained were treated with OmniSEC software (Viscotek). 2.3. PLGA–PEG–PLGA-hydrolyzable crosslinker synthesis The crosslinker synthesis is a two-step reaction comprising polyester block polymerization initiated by hydroxyl-terminated PEG chains and end polymer chain modification with polymerizable double bonds. PEG (10.07 g, 16.8 mmol), DL-lactide (7.26 g, 50.4 mmol), glycolide (5.86 g, 50.5 mmol) and stannous 2-ethylhexanoate (128 mg, 0.3 mmol) were added to a dry Schlenk tube and subjected to several vacuum–argon cycles. The Schlenk tube was then heated at 115 °C for 20 h under argon atmosphere with continuous magnetic stirring. After the reaction had been completed, the polymer was cooled and then dissolved in 40 ml of dichloromethane and precipitated twice, first in a 1 l equivolumic mixture of diethyl ether and petroleum ether and then in petroleum ether, to remove any traces of unreacted monomer. Purified polymer was dried under a vacuum at room temperature and characterized by 1H NMR. The final product is a translucent gel (yield 95%).
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1
H NMR (CDCl3) d (ppm): 5.06–5.35 (m, CH, LA), 4.56–4.93 (m, CH2, GA), 4.2–4.45 (m, CO–O–CH2 of PEG, CH or CH2 of the last LA or GA units), 3.36–3.81 (m, CH2 of PEG), 1.37–1.71 (m, CH3, LA). The peak corresponding to the CH2 of ethylene glycolide was taken as a reference for the calculation of the polymerization degree of both lactide and glycolide units. PLGA–PEG–PLGA copolymer (3 g, 2.2 mmol) was added to a Schlenk tube and dissolved in 30 ml of degassed ethyl acetate. The reaction mixture was cooled at 0 °C in a glass bath and, after 5 min of gentle stirring, triethylamine (6 eq.) was added dropwise under an argon flow. Methacrylic anhydride (6 eq.) was then added dropwise, also under the argon flow. The final solution was stirred for 1 h at 0 °C, then heated at 80 °C for 8 h. The mixture was precipitated three times in petroleum ether to remove excess methacrylic anhydride and triethylamine. Purified polymer was dried under a vacuum at room temperature and characterized by 1H NMR. The final product is a light yellow gel (yield 95%). 1 H NMR (CDCl3) d (ppm): 6.13–6.29 ppm (m, CH@), 5.55– 5.7 ppm (m, CH@), 5.06–5.35 ppm (m, CH, LA), 4.56–4.93 ppm (m, CH2, GA), 4.31 (m, CO–O–CH2, PEG), 3.36–3.81 ppm (m, PEG), 1.8–2.05 ppm (m, CH3 methacrylate), 1.37–1.71 ppm (m, CH3, LA). The functionalization degree was calculated using the peak corresponding to the CH2 of ethylene glycolide as a reference and the peak of the CH of the methacrylic group. 2.4. Linear polymers and gels synthesis In a typical linear polymer synthesis, PEGMA (2 g, 6.6 mmol), MDO (85 mg, 0.7 mmol) and 1-hexanethiol (28 ll, 0.2 mmol) were added to a vial and dissolved in 2 g of toluene. The vial was heated at 80 °C for 8 h under gentle magnetic stirring. After the reaction had been completed, 10 ml of dichloromethane was added to the vial and the reactive medium was precipitated in petroleum ether to remove any traces of unreacted monomer. Purified polymer was dried under a vacuum at room temperature, then characterized by 1 H NMR and SEC. The final product was a slightly white gel (yield 98%). 1 H NMR (CDCl3) d (ppm): 3.8–4.3 (m, CO–O–CH2, MDO and PEGMA), 3.5–3.8 (m, CH2, PEGMA), 3.3–3.5 (s, CH3, PEGMA) and 0.7–2.2 (m, CH2–CO and CH2, MDO and CH2–C(CH3)–, PEGMA). The MDO content was calculated using the peak corresponding to the CH3 of PEGMA as a reference and the peak of CO–O–CH2 of the MDO and PEGMA blocks. For gels synthesis, the PLGA–PEG–PLGA crosslinker was added to the reactive medium at 5 mol.%. After the reaction had been completed, gels were washed with acetone and water to remove any traces of unreactive monomer. 2.5. Microspheres preparation The MS process parameters (i.e. the oil-to-water ratio, the monomer mass concentration of the organic phase, the reaction temperature and the stirring speed) were selected to obtain MS with a diameter between 300 and 500 lm and kept constant along this study. In a typical experiment, an aqueous solution (111 ml) containing PVA (0.5 wt.%) and NaCl (3 wt.%) was added to a 0.25 l reactor. The dispersed phase, containing PEGMA (5.38 g, 18 mmol), PLGA–PEG– PLGA crosslinker (1.816 g, 1.2 mmol), MA (206 mg, 2.4 mmol), MDO (273 mg, 2.4 mmol), 1-hexanethiol (76 ll, 0.54 mmol) and AIBN (42 mg, 0.25 mmol) solubilized in 6.15 g of toluene, was degassed by nitrogen bubbling for 15 min. The solution was added to the aqueous phase at 80 °C and stirred at 180 rpm by using an Inox impeller for 8 h. The MS were collected by filtration on a 100 lm sieve and washed extensively with acetone and water. They were then sieved with decreasing sizes of sieve (800, 500, 300 and
100 lm). All MS were freeze dried immediately after purification. Dry MS pellets were then sterilized by beta-irradiation (Ionisos, France) and stored at 20 °C until use. The mass yield of the MS synthesis is 78%. 2.6. Hydration kinetics Dry pellets of sterilized MS (300–500 lm) were loaded onto a glass slide using a plastic tip and pictures were taken with microscope (Leitz DIAPLAN) (magnification 2.5). The dry pellets were then hydrated by adding saline solution (Versol, Laboratoire Aguettant) and, after 5 min, pictures of the MS were taken with a microscope. The wet MS were transferred to a 15 ml tube with 1 ml of saline and incubated at room temperature with occasional shaking. After 30 min and 3 h of incubation, MS were removed from the tube to a glass slide and pictures were taken. The same experiment of MS swelling was performed in a 1/1 mixture of water with non-ionic contrast medium (Omnipaque, GE). The diameters of MS (521 dry MS, 1614 MS swollen in saline and 1627 MS swollen in saline/Omnipaque mixture, respectively) were determined using the ImageJ software. 2.7. Swelling evaluation In triplicate, 1 ml of wet MS was placed in pre-weighted 15 ml polypropylene vials before freeze-drying. The dry MS weight was determined. MS were then incubated for 10 min in distilled water and the supernatant and interstitial water between the MS were removed by aspiration with a pipette tip. The tube was weighted again and the amount of absorbed water calculated. The same procedure was repeated with phosphate buffer (0.1 M phosphate-buffered saline (PBS) with 0.9% NaCl and 0.02% NaN3, pH 7.4). The solvent taken up by MS was calculated according to the formula (Ws Wd)/Wd, where Ws was the weight of the swollen MS and Wd was the weight of the dry MS. 2.8. MS injectability To test their mechanical compliance, MS were dispersed in a solution composed of 50 vol.% saline solution (Versol, Laboratoire Aguettant) and 50 vol.% contrast medium (Omnipaque, GE), then injected via a microcatheter (EV3, Echelon 10, inner diameter 430 lm) in a vial. 2.9. Doxorubicin loading on MS Aliquots of 0.1 ml of MS, either degradable MS or DC-beadsTM (Biocompatibles, Farnham, UK), were incubated with 3.5 mg of lyophilized doxorubicin (Adriblastin, Pfizer) reconstituted in water (2.5 mg ml1). Next, 50 ll of sodium hydrogen carbonate 1.4% (Fresenius Kabi, France) was added to the tubes containing degradable MS to slightly elevate the pH of the solution. Tubes were then incubated for 1 h at room temperature under agitation. The doxorubicin remaining in the supernatant after 1 h was quantified by optical density reading at 492 nm in a 96-well microplate reader. The loaded dose was calculated by subtracting the final amount of doxorubicin from the initial amount. The loading efficiency was calculated by the following equation: Loading efficiency ¼
Doxorubicin in feed doxorubicin in supernatant 100 Doxorubicin in feed
2.10. In vitro degradation In triplicate, wet MS were placed in pre-weighted 15 ml polypropylene tubes before freeze-drying. Dry MS weights were determined and phosphate buffer was added to make a
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concentration of 10 mg ml1 MS. The tubes were then shaken horizontally at 200 rpm at 37 °C in an orbital shaker oven (IKA, KS4000i control). The samples were taken out after 0, 6, 15, 24 and 48 h. After MS decantation, the pH values of the degradation media were collected (Mettler Toledo, type M8220). The media were then removed and the MS were rinsed (3 10 ml) with deionized water before freeze-drying. Tubes were re-weighed to determine the weight loss using the following equation: weight loss (%) = (W0 Wt)/W0 100, where W0 and Wt are the dry weight of the sample before and after degradation, respectively. The reported weight loss was the average of three samples.
2.11. Linear polymers, gels and MS degradation products The hydrolytic degradation of linear polymers, gels or MS was performed as follows: 150 mg of linear polymers, 150 mg of gels or 1 ml of wet MS sediment was added to a 15 ml Falcon tube and made up to 10 ml with 0.1 M NaOH solution. The solutions were mechanically stirred for 48 h at 37 °C in an incubator (176 rpm) and dialyzed against deionized water for 48 h to remove salt and low-molecular-weight degradation products (dialysis membrane molecular weight cut off: 100–500 Da). 1 H NMR analysis of the MS degradation products was performed in dimethylsulfoxide-d6 on lyophilized MS degradation products (see the figure in the Supplementary data). To solubilize the degradation products in the SEC solvent (THF), they were chemically modified through the methylation of carboxylic groups using trimethylsilydiazomethane. For this, 10 ml of dialysate was added to a 100 ml round-bottom flask togwther with 40 ml of THF. TMS solution (roughly 1 ml) was added dropwise to the solution, which turned yellow. The solution was stirred overnight at 20 °C. After the completion of the reaction, any excess THF, hexane and TMS was removed by rotary evaporation. Finally, the methylated polymer was lyophilized prior to its solubilization in the SEC solvent for analysis.
2.12. Cytotoxicity of microsphere extracts Cytotoxicity of MS was analyzed using extracts of MS prepared in cell culture medium. Briefly, mouse fibroblasts (L929) cultures were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 2 mM L-glutamine, 50 lg ml1 streptomycin and 50 units ml1 penicillin in a CO2 incubator at 37 °C. L929 cells harvesting was performed using trypsin–ethylenediaminetetraacetic acid (Lonza) and subcultures started in 96-well plates (NUNC) at densities of 5 103 cells well1. MS extracts were prepared in sterile tubes by adding 500 ll of MS pellets in DMEM and making up the volume to 3 ml with cell culture medium without serum. Samples were incubated at 37 °C under agitation until the MS had completely degraded. Chirurgical glove fragments (latex) and Embosphere were used as the positive and negative control of cytotoxicity, respectively. The day after cell seeding, bovine serum was added to the MS extracts and the pH was adjusted to pH 7 with 20 mM sodium hydrogen carbonate (Fresenius Kabi, France) before non-confluent fibroblasts were added (6–8 wells per condition). Extracts obtained from the chirurgical gloves and GMS were also added to mouse fibroblasts. After 72 h of culture (37 °C, 5% CO2), the medium was removed and the cells were washed with 100 ll of PBS, before the addition of 100 ll of bicinchoninic acid solution (BCA protein reagent, Sigma) containing 0.08 wt./vol.% CuSO4 and 0.05% Triton X-100. After incubation (1 h at 37 °C), absorbance was measured at 570 nm and the amount of proteins was obtained by extrapolation from the standard curve using bovine serum albumin.
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2.13. Contact toxicity In 24-wells plates, L929 cells were seeded at 50,000 cells well1 in cell culture medium (see above). The day after seeding, the medium was removed and replaced with 0.9 ml of culture medium containing 100 mM HEPES. Then 100 ll of sterilized microsphere sediment was added (in duplicate) and the cells were co-cultured with MS for 1 week (37 °C, 5% CO2). Pieces of latex gloves and non-degradable GMS were used as the positive and negative control of cytotoxicity, respectively. At the end of culture, pH of culture medium was estimated using pH indicator paper and presence of non-degradable MS was checked. The amount of total cell proteins was determined by a BCA assay. Briefly, the medium is removed and the cells are washed with PBS, then lysed with 300 ll of PBS containing 0.05% Triton X-100 and 1 unit of DNAse I for 1 h at 37 °C. The proteins are determined in triplicate using 20 ll of cell lysate.
2.14. Subcutaneous implantation in rabbit Experimental procedures were performed at the Center of Research in Interventional Radiology (Cr2i/APHP/National Institute of Agronomic Research INRA, Jouy-en-Josas, France). The study protocol was approved by the Institutional Animal Care and Use Committee of the Center and was conducted according to European Community Rules of Animal Care. Different degradable MS (PEG–PLGA, PEG–PLGA–MA and PEG– PLGA–MA–MDO) were implanted into the back skin of white New Zealand rabbits (n = 2 per group). Briefly, anesthesia was induced by 5% isoflurane/95% oxygen through a mask. The implantation sites were shaved and tattooed. A 0.6 ml volume of microspheres swelled in saline was injected through a 14G needle in six 4 cm long parallel rails per rabbit to avoid a cluster effect. Rabbits were then sacrificed on day 2 or 7. In addition, four rabbits were injected with saline only (sham) as controls and sacrificed on day 2 or 7. At sacrifice, the skin was explanted according to the tattooed marks, attached to a plate and fixed in 10% neutral buffered formalin for at least 24 h. For each rabbit, a 4 mm thick tissue slice was cut transversally from each of the six implant rails. The samples were dehydrated in a series of alcohols, set in xylene and embedded in paraffin. Sections 3–4 lm thick were cut and mounted on a microscope slide. After rehydration in a series of alcohols, sections were stained with hematein–eosin–saffron. Tissue reaction and material degradation were assessed by histology. The frequency of observation of amounts of (almost degraded) MS in the histological slides was calculated. The main organs were harvested at sacrifice and checked by a vet and a pathologist for abnormalities that would suggest systemic toxicity.
3. Results 3.1. Formulation and preparation of hydrolyzable microspheres A hydrolyzable crosslinker, PLGA–PEG–PLGA dimethacrylate, was prepared via an established protocol [22]. Precursor was first synthesized by ring-opening polymerization of lactide and glycolide using Mw 600 PEG as the initiator. The composition of the oligomer obtained was found to be similar to the corresponding monomer feed ratio. The hydroxy-terminated precursor was then allowed to react with methacrylic anhydride to form crosslinkable polyesters. The complete conversion of the terminal OH groups by the methacrylate group was confirmed by 1H NMR.
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A series of microspheres were then prepared by suspension polymerization using 5 mol.% hydrolyzable crosslinker and PEGMA as the main monomer (Fig. 1). Co-monomers such as MA and MDO were incorporated to improve the degradation characteristics of the microspheres: MA allows drug loading and improves water uptake when ionized; MDO decreases the molecular weight of the residual polymer chains after hydrolysis. The cyclic monomer MDO is able to (co)polymerize through a free radical mechanism either by a ring-opening reaction to form an ester in the main chain or by a vinylic addition reaction at the double bond with retention of the ring structure [23]. Prior to its incorporation into the microspheres, the MDO was copolymerized with PEGMA at different feed ratios, using AIBN at 80 °C as the initiator. In all cases, the molar ratio of PEGMA/MDO, as determined by 1H NMR, was higher in the linear copolymers than in the initial feed (Table 1). No peak around 100–110 ppm, corresponding to the acetal carbon of the ring-retained structure of MDO, was evidenced in the 13C NMR spectra of the copolymers. Thus, copolymerization of MDO with PEGMA in this condition led to quantitative ring opening of MDO. To determine the influence of the introduction of MDO on the reduction of the molar mass, gels of MDO/PEGMA containing 5 mol.% hydrolyzable crosslinker with increasing proportions of MDO in the monomers mixtures were synthesized in toluene at 80 °C using AIBN (Table 1). The number-average molecular weights of the remaining polymer chains after degradation of the gels under alkaline conditions rapidly decreased with increasing molar feed ratio of MDO to PEGMA. At 10 mol.% MDO in the monomers mixture, the molecular weight (Mn = 24,000 g mol1) was far below the threshold of 50,000 g mol1. Above 20 mol.% MDO, it was not possible to determine the molecular weight precisely, as they were similar to or below the permeation limit of the columns. Microspheres were prepared from different monomer compositions (Table 2). Whatever the formulations used, the resulting MS were all spherical in shape (Fig. 2), with a diameter mainly in the range 300–500 lm. The FTIR spectra of the hydrolyzable crosslinker and the PEG–PLGA MS are shown in Fig. 3. The characteristic absorption bands of C = CH2 stretch (1634 and 810 cm1) from the crosslinker disappeared in the PEG–PLGA microsphere due to their consumption during the suspension polymerization. Meanwhile, the absorption peak of the saturated
ester groups (1736 cm1) appeared as a shoulder in the IR spectrum of the MS. A strong absorbance was seen at 1755 cm1 in the crosslinker, indicating the presence of ester stretch in PLA and PGA units. This absorbance remained after PEG-PLGA formation of the MS, indicating the good incorporation of the hydrolyzable crosslinker. The peak at 1726 cm1 in the MS spectrum was attributed to the carbonyl absorption of the saturated ester groups of PEGMA.
3.2. Swelling behaviour The microsphere swelling ratio was measured in water (pH 6) and PBS (pH 7.4) (Table 2). In both media, the presence of 10 mol.% MA increased the swelling ratio to approximately 20% in water and 50% in PBS. In PBS at pH 7.4, the carboxylic groups of the MA units were more dissociated than in water, resulting in swelling of the MS. Incorporation of MDO into the polymeric network induced a slight decrease in the swelling ratio. Despite its hydrophobic structure, the low level of MDO incorporation (around 7 mol.% according to NMR) did not modify the swelling properties of the MS. After the lyophilization and sterilization steps, the MS regained their initial swelling state rapidly (see the figure in the Supplementary data). The diameter of dry MS (259 ± 17 lm) increased to 410 ± 26 and 407 ± 23 lm after 5 min of incubation in saline or in the saline:/Omnipaque mixture, respectively. After 3 h at room temperature the diameter of the MS in saline did not change (405 ± 27 lm). In the saline/contrast medium mixture, the diameter increased slightly (429 ± 28 lm at 30 min) and did not change up to 3 h (431 ± 29 lm).
3.3. Injectability All the MS were easily injected through a echelon™ 10 microcatheter (inner diameter 430 lm). The force needed for manual injection was similar to that needed for injection of 300–500 lm Ò Embosphere under the same conditions. No aggregation or catheter blockage was found. The MS recovered their spherical shape at the exit of the catheter without residual deformation.
Fig. 1. Reaction scheme for the synthesis of PEG–PLGA microspheres.
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S. Louguet et al. / Acta Biomaterialia 10 (2014) 1194–1205 Table 1 Characterization of linear polymers and crosslinked gels containing increasing proportions of MDO.
a
MDO:PEGMA (mol.%)
Crosslinker (mol.%)
PEGMA:MDO (1H NMR)
Mn (g mol1)
Mn after hydrolysis (g mol1)a
Mw/Mn
Linear Linear Linear
10:90 20:80 30:70
– – –
7:93 13:87 20:80
24 000 28 100 29 200
14,000 8000 6700
1.6 1.7 1.6
Gel Gel Gel Gel Gel
0:95 10:85 20:75 30:65 40:55
5 5 5 5 5
ND ND ND ND ND
– – – – –
61,000 24,000
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Poly(ethylene glycol) methacrylate hydrolyzable microspheres for transient vascular embolization Stéphanie Louguet a, Valentin Verret a,b, Laurent Bédouet a, Emeline Servais a, Florentina Pascale b, Michel Wassef c, Denis Labarre d,e, Alexandre Laurent c,f, Laurence Moine d,e,⇑ a
Occlugel S.A.S., 12 Rue Charles de Gaulle, 78350 Jouy en Josas, France Archimmed S.A.R.L., 12 Rue Charles de Gaulle, 78350 Jouy en Josas, France AP-HP hôpital Lariboisière, Department of Pathology, University of Paris 7 – Denis Diderot, Faculty of Medicine, 2 rue Ambroise Paré, 75010 Paris, France d Université Paris-Sud, Institut Galien Paris-Sud, LabEx LERMIT, Faculté de Pharmacie, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France e CNRS UMR 8612, Institut Galien Paris-Sud, LabEx LERMIT, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France f ‘‘Laboratoire Matières et Systèmes Complexes’’, CNRS 7057, University of Paris 7, Bâtiment Condorcet, 10 rue Alice Domon et Léonie Duquet, 75205 Paris Cedex 13, France b c
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 6 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Embolization Microsphere Degradable Hydrolyzable crosslinker PEG-based hydrogel
a b s t r a c t Poly(ethylene glycol) methacrylate (PEGMA) hydrolyzable microspheres intended for biomedical applications were readily prepared from poly(lactide-co-glycolide) (PLGA)–poly(ethylene glycol) (PEG)–PLGA crosslinker and PEGMA as a monomer using a suspension polymerization process. Additional co-monomers, methacrylic acid and 2-methylene-1,3-dioxepane (MDO), were incorporated into the initial formulation to improve the properties of the microspheres. All synthesized microspheres were spherical in shape, calibrated in the 300–500 lm range, swelled in phosphate-buffered saline (PBS) and easily injectable through a microcatheter. Hydrolytic degradation experiments performed in PBS at 37 °C showed that all of the formulations tested were totally degraded in less than 2 days. The resulting degradation products were a mixture of low-molecular-weight compounds (PEG, lactic and glycolic acids) and water-soluble polymethacrylate chains having molecular weights below the threshold for renal filtration of 50 kg mol1 for the microspheres containing MDO. Both the microspheres and the degradation products were determined to exhibit minimal cytotoxicity against L929 fibroblasts. Additionally, in vivo implantation in a subcutaneous rabbit model supported the in vitro results of a rapid degradation rate of microspheres and provided only a mild and transient inflammatory reaction comparable to that of the control group. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Embolization, i.e. occlusion of arteries feeding organs with injected biomaterials, is a technique used for the treatment of various pathologies such as tumors, arterio-venous shunts and hemorrhages. It is commonly achieved with particles, which can be either degradable or non-degradable. The degradable particles are preferred when a durable vessel occlusion is not required, for example in uterine fibroids that are very sensitive to ischemia, in bleedings where the blood leak can be stopped if the flow is blocked for a few hours, or in some tumors, specially malignant ones, when embolization has to be repeated in the same feeder arteries. Non-degradable particles have evolved considerably in the last few decades. The irregular poly(vinylalcohol) (PVA) particles of the
⇑ Corresponding author at: Université Paris-Sud, Institut Galien Paris-Sud, LabEx LERMIT, Faculté de Pharmacie, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France. E-mail address: [email protected] (L. Moine).
1970s, which were poorly calibrated, aggregated proximally in clusters and gave a chronic inflammatory response [1–3], have been replaced by smooth, biocompatible and calibrated microspheres (MS). They drastically changed the conditions of embolization, since the radiologist could adapt the size of the MS to the size of the vessels to allow precise targeting and accurate devascularization. These MS have since been chemically grafted, to be loaded ionically with cationic drugs, and have thereby become actual drug delivery systems for local chemotherapy in tumor embolization [4,5]. By comparison, the evolution of the degradable particles has been very slow. Porcine sponge particles (GSP) were used at the very beginning of embolization in the 1970s and are still commonly used today. The enzymatic degradation of GSP is highly variable over time, from 3 weeks to 4 months [6–10], and is accompanied by a chronic inflammatory response and a vessel remodelling process [6,9], so that functional recanalization cannot be assured [11,12].
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.11.028
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It is only very recently that two types of degradable MS have become available for embolization. However, their severe limitations hamper their competitiveness with non-degradable MS. Starch MS (Embocept™, Pharmacept) have been proposed to enhance drug diffusion in tumoral tissues when coadministered with chemotherapeutic drugs [13–15]. However, their small diameter (500 lm). Collagen-coated poly(lactide-co-glycolide) (PLGA) MS (Occlusin™500, IMBiotechnologies) are also available, but in only one small size (150–212 lm). Further, their in vivo degradation requires several months and is accompanied by an inflammatory and fibrotic reaction [16]. We have therefore conceived a degradable microsphere that encompasses the useful properties of non-degradable MS. First, we aimed to make a degradable microsphere which temporary occludes the vessel between a few hours and a few days, which is sufficient to achieve an ischemia of tumors (in the case of fibroids), a vessel repair (in the case of bleeding) or the delivery of a drug to tumors (in the case of chemoembolization). Second, this biomaterial should, after its degradation, be eliminated quickly, before the onset of a chronic inflammatory response and vessel wall remodelling [l2]. Third, the MS must be calibrated in several size ranges, between 100 and 1000 lm. Fourth, the MS should be easily suspended in physiological solutions and contrast medium. Fifth, the material must be soft and elastic enough to be injected via microcatheters of diameters smaller than the MS, and to recover their size and shape upon exit [17]. Sixth, the MS must be loadable with drugs for transient chemoembolization procedures. To fulfill the above-listed requirements, we have successfully developed a tunable and degradable MS that combines poly(ethylene glycol) methacrylate (PEGMA) and a hydrolyzable crosslinker (PLGA–poly(ethylene glycol) (PEG)–PLGA). PEGMA was selected as the monomer because of its amphiphilic nature, which results from its water-soluble PEG side chain and its hydrophobic methacrylate group. The amphiphilic nature of PEGMA makes possible mixing it directly with the hydrolyzable crosslinker in hydrocarbon phase to perform the polymerization by direct oil in water suspension process for making microspheres. The hydrolyzable crosslinker was designed to achieve rapid degradation time. An anionic co-monomer, methacrylic acid (MA) was combined to the PEGMA monomer and the crosslinker to adjust the water uptake and to allow the ionic loading of drugs. A large part of the strategy was devoted to the biocompatibility of the material. First, the initial components were chosen to generate water-soluble macromolecules after hydrolysis in order to avoid any accumulation in the organism, which might lead to inflammatory reactions [18]. Second, additional ester linkages from a cyclic ketene acetal monomer, 2-methylene-1,3-dioxepane (MDO), were incorporated into the hydrogel backbone to shorten the size of the polymer degradation products (below 50 kg mol1), facilitating their renal elimination [19,20]. We report here the feasibility of preparing suitable degradable microspheres for embolization with the selected components (hydrolyzable crosslinker, PEGMA, MA and MDO). The impact of each component on the degradation time and on the size of the degradation products was evaluated. The hydrolytic degradation of the different formulations was characterized and the effect of degradation products on the biocompatibility was studied by two different cytotoxic tests. Subcutaneous implantations were then performed in rabbits to assess the in vivo degradation of MS and the local inflammatory reaction.
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2. Material and methods 2.1. Materials PEGMA (Mn = 300 g mol1), stannous octanoate, triethylamine, methacrylic anhydride, 88% hydrolyzed PVA, glycolide, 1-hexanethiol and polyethylene glycol (Mw = 600 g mol1) (PEG) were purchased from Aldrich (St Quentin Fallavier, France). 2,20 -Azobisisobutyronitrile (AIBN), used as a polymerization initiator, and trimethylsilyldiazomethane (2 M in hexane; TMS) were obtained from Acros Organic (Geel, Belgium). D,L-Lactide was obtained from Biovalley (Marne la Vallée, France). MDO was synthesized according to the procedure published in Ref. [21]. Doxorubicin hydrochloride (Adriblastin) was purchased from Pfizer (France). Embosphere (non-degradable gelatin microsphere; GMS) was purchased from Biosphere Medical (Roissy, France). DC-beadsTM microspheres were purchased from Biocompatibles (Farnham, UK). All reagents were used without any further purification. Analytical grade solvents were supplied by Carlo Erba (Val de Rueil, France). 2.2. Instrumentation 2.2.1. Nuclear magnetic resonance (NMR) Products were analyzed by 1H NMR spectroscopy using a Bruker DPX300 FT NMR spectrometer, with the solvent peak as a reference. 2.2.2. Fourier transform infrared spectroscopy (FTIR) FTIR was used to characterized the chemical structure of polymers and MS. Spectra were recorded on a Spectrum One Perkin Elmer infrared spectrometer equipped with a diamond attenuated total reflectance module in a range of 500–4000 cm1. One hundred interferograms were averaged per spectrum at a resolution of 2 cm1. 2.2.3. Size exclusion chromatography (SEC) The molecular weight and molecular weight distribution of polymers were measured by SEC at 30 °C on a system equipped with a guard column and two GMH HRm columns (Viscotek) with tetrahydrofuran (THF) as the eluent at 1 ml min1. A differential refractometer and a double SEC detector (model 270 Dual, Viscotek) with RALS and viscosimeter in series were used to analyze samples. The data obtained were treated with OmniSEC software (Viscotek). 2.3. PLGA–PEG–PLGA-hydrolyzable crosslinker synthesis The crosslinker synthesis is a two-step reaction comprising polyester block polymerization initiated by hydroxyl-terminated PEG chains and end polymer chain modification with polymerizable double bonds. PEG (10.07 g, 16.8 mmol), DL-lactide (7.26 g, 50.4 mmol), glycolide (5.86 g, 50.5 mmol) and stannous 2-ethylhexanoate (128 mg, 0.3 mmol) were added to a dry Schlenk tube and subjected to several vacuum–argon cycles. The Schlenk tube was then heated at 115 °C for 20 h under argon atmosphere with continuous magnetic stirring. After the reaction had been completed, the polymer was cooled and then dissolved in 40 ml of dichloromethane and precipitated twice, first in a 1 l equivolumic mixture of diethyl ether and petroleum ether and then in petroleum ether, to remove any traces of unreacted monomer. Purified polymer was dried under a vacuum at room temperature and characterized by 1H NMR. The final product is a translucent gel (yield 95%).
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1
H NMR (CDCl3) d (ppm): 5.06–5.35 (m, CH, LA), 4.56–4.93 (m, CH2, GA), 4.2–4.45 (m, CO–O–CH2 of PEG, CH or CH2 of the last LA or GA units), 3.36–3.81 (m, CH2 of PEG), 1.37–1.71 (m, CH3, LA). The peak corresponding to the CH2 of ethylene glycolide was taken as a reference for the calculation of the polymerization degree of both lactide and glycolide units. PLGA–PEG–PLGA copolymer (3 g, 2.2 mmol) was added to a Schlenk tube and dissolved in 30 ml of degassed ethyl acetate. The reaction mixture was cooled at 0 °C in a glass bath and, after 5 min of gentle stirring, triethylamine (6 eq.) was added dropwise under an argon flow. Methacrylic anhydride (6 eq.) was then added dropwise, also under the argon flow. The final solution was stirred for 1 h at 0 °C, then heated at 80 °C for 8 h. The mixture was precipitated three times in petroleum ether to remove excess methacrylic anhydride and triethylamine. Purified polymer was dried under a vacuum at room temperature and characterized by 1H NMR. The final product is a light yellow gel (yield 95%). 1 H NMR (CDCl3) d (ppm): 6.13–6.29 ppm (m, CH@), 5.55– 5.7 ppm (m, CH@), 5.06–5.35 ppm (m, CH, LA), 4.56–4.93 ppm (m, CH2, GA), 4.31 (m, CO–O–CH2, PEG), 3.36–3.81 ppm (m, PEG), 1.8–2.05 ppm (m, CH3 methacrylate), 1.37–1.71 ppm (m, CH3, LA). The functionalization degree was calculated using the peak corresponding to the CH2 of ethylene glycolide as a reference and the peak of the CH of the methacrylic group. 2.4. Linear polymers and gels synthesis In a typical linear polymer synthesis, PEGMA (2 g, 6.6 mmol), MDO (85 mg, 0.7 mmol) and 1-hexanethiol (28 ll, 0.2 mmol) were added to a vial and dissolved in 2 g of toluene. The vial was heated at 80 °C for 8 h under gentle magnetic stirring. After the reaction had been completed, 10 ml of dichloromethane was added to the vial and the reactive medium was precipitated in petroleum ether to remove any traces of unreacted monomer. Purified polymer was dried under a vacuum at room temperature, then characterized by 1 H NMR and SEC. The final product was a slightly white gel (yield 98%). 1 H NMR (CDCl3) d (ppm): 3.8–4.3 (m, CO–O–CH2, MDO and PEGMA), 3.5–3.8 (m, CH2, PEGMA), 3.3–3.5 (s, CH3, PEGMA) and 0.7–2.2 (m, CH2–CO and CH2, MDO and CH2–C(CH3)–, PEGMA). The MDO content was calculated using the peak corresponding to the CH3 of PEGMA as a reference and the peak of CO–O–CH2 of the MDO and PEGMA blocks. For gels synthesis, the PLGA–PEG–PLGA crosslinker was added to the reactive medium at 5 mol.%. After the reaction had been completed, gels were washed with acetone and water to remove any traces of unreactive monomer. 2.5. Microspheres preparation The MS process parameters (i.e. the oil-to-water ratio, the monomer mass concentration of the organic phase, the reaction temperature and the stirring speed) were selected to obtain MS with a diameter between 300 and 500 lm and kept constant along this study. In a typical experiment, an aqueous solution (111 ml) containing PVA (0.5 wt.%) and NaCl (3 wt.%) was added to a 0.25 l reactor. The dispersed phase, containing PEGMA (5.38 g, 18 mmol), PLGA–PEG– PLGA crosslinker (1.816 g, 1.2 mmol), MA (206 mg, 2.4 mmol), MDO (273 mg, 2.4 mmol), 1-hexanethiol (76 ll, 0.54 mmol) and AIBN (42 mg, 0.25 mmol) solubilized in 6.15 g of toluene, was degassed by nitrogen bubbling for 15 min. The solution was added to the aqueous phase at 80 °C and stirred at 180 rpm by using an Inox impeller for 8 h. The MS were collected by filtration on a 100 lm sieve and washed extensively with acetone and water. They were then sieved with decreasing sizes of sieve (800, 500, 300 and
100 lm). All MS were freeze dried immediately after purification. Dry MS pellets were then sterilized by beta-irradiation (Ionisos, France) and stored at 20 °C until use. The mass yield of the MS synthesis is 78%. 2.6. Hydration kinetics Dry pellets of sterilized MS (300–500 lm) were loaded onto a glass slide using a plastic tip and pictures were taken with microscope (Leitz DIAPLAN) (magnification 2.5). The dry pellets were then hydrated by adding saline solution (Versol, Laboratoire Aguettant) and, after 5 min, pictures of the MS were taken with a microscope. The wet MS were transferred to a 15 ml tube with 1 ml of saline and incubated at room temperature with occasional shaking. After 30 min and 3 h of incubation, MS were removed from the tube to a glass slide and pictures were taken. The same experiment of MS swelling was performed in a 1/1 mixture of water with non-ionic contrast medium (Omnipaque, GE). The diameters of MS (521 dry MS, 1614 MS swollen in saline and 1627 MS swollen in saline/Omnipaque mixture, respectively) were determined using the ImageJ software. 2.7. Swelling evaluation In triplicate, 1 ml of wet MS was placed in pre-weighted 15 ml polypropylene vials before freeze-drying. The dry MS weight was determined. MS were then incubated for 10 min in distilled water and the supernatant and interstitial water between the MS were removed by aspiration with a pipette tip. The tube was weighted again and the amount of absorbed water calculated. The same procedure was repeated with phosphate buffer (0.1 M phosphate-buffered saline (PBS) with 0.9% NaCl and 0.02% NaN3, pH 7.4). The solvent taken up by MS was calculated according to the formula (Ws Wd)/Wd, where Ws was the weight of the swollen MS and Wd was the weight of the dry MS. 2.8. MS injectability To test their mechanical compliance, MS were dispersed in a solution composed of 50 vol.% saline solution (Versol, Laboratoire Aguettant) and 50 vol.% contrast medium (Omnipaque, GE), then injected via a microcatheter (EV3, Echelon 10, inner diameter 430 lm) in a vial. 2.9. Doxorubicin loading on MS Aliquots of 0.1 ml of MS, either degradable MS or DC-beadsTM (Biocompatibles, Farnham, UK), were incubated with 3.5 mg of lyophilized doxorubicin (Adriblastin, Pfizer) reconstituted in water (2.5 mg ml1). Next, 50 ll of sodium hydrogen carbonate 1.4% (Fresenius Kabi, France) was added to the tubes containing degradable MS to slightly elevate the pH of the solution. Tubes were then incubated for 1 h at room temperature under agitation. The doxorubicin remaining in the supernatant after 1 h was quantified by optical density reading at 492 nm in a 96-well microplate reader. The loaded dose was calculated by subtracting the final amount of doxorubicin from the initial amount. The loading efficiency was calculated by the following equation: Loading efficiency ¼
Doxorubicin in feed doxorubicin in supernatant 100 Doxorubicin in feed
2.10. In vitro degradation In triplicate, wet MS were placed in pre-weighted 15 ml polypropylene tubes before freeze-drying. Dry MS weights were determined and phosphate buffer was added to make a
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concentration of 10 mg ml1 MS. The tubes were then shaken horizontally at 200 rpm at 37 °C in an orbital shaker oven (IKA, KS4000i control). The samples were taken out after 0, 6, 15, 24 and 48 h. After MS decantation, the pH values of the degradation media were collected (Mettler Toledo, type M8220). The media were then removed and the MS were rinsed (3 10 ml) with deionized water before freeze-drying. Tubes were re-weighed to determine the weight loss using the following equation: weight loss (%) = (W0 Wt)/W0 100, where W0 and Wt are the dry weight of the sample before and after degradation, respectively. The reported weight loss was the average of three samples.
2.11. Linear polymers, gels and MS degradation products The hydrolytic degradation of linear polymers, gels or MS was performed as follows: 150 mg of linear polymers, 150 mg of gels or 1 ml of wet MS sediment was added to a 15 ml Falcon tube and made up to 10 ml with 0.1 M NaOH solution. The solutions were mechanically stirred for 48 h at 37 °C in an incubator (176 rpm) and dialyzed against deionized water for 48 h to remove salt and low-molecular-weight degradation products (dialysis membrane molecular weight cut off: 100–500 Da). 1 H NMR analysis of the MS degradation products was performed in dimethylsulfoxide-d6 on lyophilized MS degradation products (see the figure in the Supplementary data). To solubilize the degradation products in the SEC solvent (THF), they were chemically modified through the methylation of carboxylic groups using trimethylsilydiazomethane. For this, 10 ml of dialysate was added to a 100 ml round-bottom flask togwther with 40 ml of THF. TMS solution (roughly 1 ml) was added dropwise to the solution, which turned yellow. The solution was stirred overnight at 20 °C. After the completion of the reaction, any excess THF, hexane and TMS was removed by rotary evaporation. Finally, the methylated polymer was lyophilized prior to its solubilization in the SEC solvent for analysis.
2.12. Cytotoxicity of microsphere extracts Cytotoxicity of MS was analyzed using extracts of MS prepared in cell culture medium. Briefly, mouse fibroblasts (L929) cultures were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 2 mM L-glutamine, 50 lg ml1 streptomycin and 50 units ml1 penicillin in a CO2 incubator at 37 °C. L929 cells harvesting was performed using trypsin–ethylenediaminetetraacetic acid (Lonza) and subcultures started in 96-well plates (NUNC) at densities of 5 103 cells well1. MS extracts were prepared in sterile tubes by adding 500 ll of MS pellets in DMEM and making up the volume to 3 ml with cell culture medium without serum. Samples were incubated at 37 °C under agitation until the MS had completely degraded. Chirurgical glove fragments (latex) and Embosphere were used as the positive and negative control of cytotoxicity, respectively. The day after cell seeding, bovine serum was added to the MS extracts and the pH was adjusted to pH 7 with 20 mM sodium hydrogen carbonate (Fresenius Kabi, France) before non-confluent fibroblasts were added (6–8 wells per condition). Extracts obtained from the chirurgical gloves and GMS were also added to mouse fibroblasts. After 72 h of culture (37 °C, 5% CO2), the medium was removed and the cells were washed with 100 ll of PBS, before the addition of 100 ll of bicinchoninic acid solution (BCA protein reagent, Sigma) containing 0.08 wt./vol.% CuSO4 and 0.05% Triton X-100. After incubation (1 h at 37 °C), absorbance was measured at 570 nm and the amount of proteins was obtained by extrapolation from the standard curve using bovine serum albumin.
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2.13. Contact toxicity In 24-wells plates, L929 cells were seeded at 50,000 cells well1 in cell culture medium (see above). The day after seeding, the medium was removed and replaced with 0.9 ml of culture medium containing 100 mM HEPES. Then 100 ll of sterilized microsphere sediment was added (in duplicate) and the cells were co-cultured with MS for 1 week (37 °C, 5% CO2). Pieces of latex gloves and non-degradable GMS were used as the positive and negative control of cytotoxicity, respectively. At the end of culture, pH of culture medium was estimated using pH indicator paper and presence of non-degradable MS was checked. The amount of total cell proteins was determined by a BCA assay. Briefly, the medium is removed and the cells are washed with PBS, then lysed with 300 ll of PBS containing 0.05% Triton X-100 and 1 unit of DNAse I for 1 h at 37 °C. The proteins are determined in triplicate using 20 ll of cell lysate.
2.14. Subcutaneous implantation in rabbit Experimental procedures were performed at the Center of Research in Interventional Radiology (Cr2i/APHP/National Institute of Agronomic Research INRA, Jouy-en-Josas, France). The study protocol was approved by the Institutional Animal Care and Use Committee of the Center and was conducted according to European Community Rules of Animal Care. Different degradable MS (PEG–PLGA, PEG–PLGA–MA and PEG– PLGA–MA–MDO) were implanted into the back skin of white New Zealand rabbits (n = 2 per group). Briefly, anesthesia was induced by 5% isoflurane/95% oxygen through a mask. The implantation sites were shaved and tattooed. A 0.6 ml volume of microspheres swelled in saline was injected through a 14G needle in six 4 cm long parallel rails per rabbit to avoid a cluster effect. Rabbits were then sacrificed on day 2 or 7. In addition, four rabbits were injected with saline only (sham) as controls and sacrificed on day 2 or 7. At sacrifice, the skin was explanted according to the tattooed marks, attached to a plate and fixed in 10% neutral buffered formalin for at least 24 h. For each rabbit, a 4 mm thick tissue slice was cut transversally from each of the six implant rails. The samples were dehydrated in a series of alcohols, set in xylene and embedded in paraffin. Sections 3–4 lm thick were cut and mounted on a microscope slide. After rehydration in a series of alcohols, sections were stained with hematein–eosin–saffron. Tissue reaction and material degradation were assessed by histology. The frequency of observation of amounts of (almost degraded) MS in the histological slides was calculated. The main organs were harvested at sacrifice and checked by a vet and a pathologist for abnormalities that would suggest systemic toxicity.
3. Results 3.1. Formulation and preparation of hydrolyzable microspheres A hydrolyzable crosslinker, PLGA–PEG–PLGA dimethacrylate, was prepared via an established protocol [22]. Precursor was first synthesized by ring-opening polymerization of lactide and glycolide using Mw 600 PEG as the initiator. The composition of the oligomer obtained was found to be similar to the corresponding monomer feed ratio. The hydroxy-terminated precursor was then allowed to react with methacrylic anhydride to form crosslinkable polyesters. The complete conversion of the terminal OH groups by the methacrylate group was confirmed by 1H NMR.
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A series of microspheres were then prepared by suspension polymerization using 5 mol.% hydrolyzable crosslinker and PEGMA as the main monomer (Fig. 1). Co-monomers such as MA and MDO were incorporated to improve the degradation characteristics of the microspheres: MA allows drug loading and improves water uptake when ionized; MDO decreases the molecular weight of the residual polymer chains after hydrolysis. The cyclic monomer MDO is able to (co)polymerize through a free radical mechanism either by a ring-opening reaction to form an ester in the main chain or by a vinylic addition reaction at the double bond with retention of the ring structure [23]. Prior to its incorporation into the microspheres, the MDO was copolymerized with PEGMA at different feed ratios, using AIBN at 80 °C as the initiator. In all cases, the molar ratio of PEGMA/MDO, as determined by 1H NMR, was higher in the linear copolymers than in the initial feed (Table 1). No peak around 100–110 ppm, corresponding to the acetal carbon of the ring-retained structure of MDO, was evidenced in the 13C NMR spectra of the copolymers. Thus, copolymerization of MDO with PEGMA in this condition led to quantitative ring opening of MDO. To determine the influence of the introduction of MDO on the reduction of the molar mass, gels of MDO/PEGMA containing 5 mol.% hydrolyzable crosslinker with increasing proportions of MDO in the monomers mixtures were synthesized in toluene at 80 °C using AIBN (Table 1). The number-average molecular weights of the remaining polymer chains after degradation of the gels under alkaline conditions rapidly decreased with increasing molar feed ratio of MDO to PEGMA. At 10 mol.% MDO in the monomers mixture, the molecular weight (Mn = 24,000 g mol1) was far below the threshold of 50,000 g mol1. Above 20 mol.% MDO, it was not possible to determine the molecular weight precisely, as they were similar to or below the permeation limit of the columns. Microspheres were prepared from different monomer compositions (Table 2). Whatever the formulations used, the resulting MS were all spherical in shape (Fig. 2), with a diameter mainly in the range 300–500 lm. The FTIR spectra of the hydrolyzable crosslinker and the PEG–PLGA MS are shown in Fig. 3. The characteristic absorption bands of C = CH2 stretch (1634 and 810 cm1) from the crosslinker disappeared in the PEG–PLGA microsphere due to their consumption during the suspension polymerization. Meanwhile, the absorption peak of the saturated
ester groups (1736 cm1) appeared as a shoulder in the IR spectrum of the MS. A strong absorbance was seen at 1755 cm1 in the crosslinker, indicating the presence of ester stretch in PLA and PGA units. This absorbance remained after PEG-PLGA formation of the MS, indicating the good incorporation of the hydrolyzable crosslinker. The peak at 1726 cm1 in the MS spectrum was attributed to the carbonyl absorption of the saturated ester groups of PEGMA.
3.2. Swelling behaviour The microsphere swelling ratio was measured in water (pH 6) and PBS (pH 7.4) (Table 2). In both media, the presence of 10 mol.% MA increased the swelling ratio to approximately 20% in water and 50% in PBS. In PBS at pH 7.4, the carboxylic groups of the MA units were more dissociated than in water, resulting in swelling of the MS. Incorporation of MDO into the polymeric network induced a slight decrease in the swelling ratio. Despite its hydrophobic structure, the low level of MDO incorporation (around 7 mol.% according to NMR) did not modify the swelling properties of the MS. After the lyophilization and sterilization steps, the MS regained their initial swelling state rapidly (see the figure in the Supplementary data). The diameter of dry MS (259 ± 17 lm) increased to 410 ± 26 and 407 ± 23 lm after 5 min of incubation in saline or in the saline:/Omnipaque mixture, respectively. After 3 h at room temperature the diameter of the MS in saline did not change (405 ± 27 lm). In the saline/contrast medium mixture, the diameter increased slightly (429 ± 28 lm at 30 min) and did not change up to 3 h (431 ± 29 lm).
3.3. Injectability All the MS were easily injected through a echelon™ 10 microcatheter (inner diameter 430 lm). The force needed for manual injection was similar to that needed for injection of 300–500 lm Ò Embosphere under the same conditions. No aggregation or catheter blockage was found. The MS recovered their spherical shape at the exit of the catheter without residual deformation.
Fig. 1. Reaction scheme for the synthesis of PEG–PLGA microspheres.
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S. Louguet et al. / Acta Biomaterialia 10 (2014) 1194–1205 Table 1 Characterization of linear polymers and crosslinked gels containing increasing proportions of MDO.
a
MDO:PEGMA (mol.%)
Crosslinker (mol.%)
PEGMA:MDO (1H NMR)
Mn (g mol1)
Mn after hydrolysis (g mol1)a
Mw/Mn
Linear Linear Linear
10:90 20:80 30:70
– – –
7:93 13:87 20:80
24 000 28 100 29 200
14,000 8000 6700
1.6 1.7 1.6
Gel Gel Gel Gel Gel
0:95 10:85 20:75 30:65 40:55
5 5 5 5 5
ND ND ND ND ND
– – – – –
61,000 24,000
