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Transcatheter embolization using degradable crosslinked hydrogels
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Biomaterials 25 (2004) 5209–5215
Transcatheter embolization using degradable crosslinked hydrogels Alexander Schwarza, Hongmin Zhanga, Annick Metcalfeb,*, Igor Salazkinb, Jean Raymondb b
a Biosphere Medical, Inc., 1050 Hingham Street, Rockland, MA 02370, USA Interventional Neuroradiology Laboratory, CHUM Research Center, Notre-Dame Hospital, Mailloux Pavilion M-8206, 1560 Sherbrooke East, Montreal, Que., Canada H2L 4M1
Received 5 December 2003; accepted 8 December 2003
Abstract Therapeutic embolization is the selective transcatheter blockage of blood vessels or diseased vascular structures. The majority of current embolization materials in clinical use are permanent. There are clinical situations however, in which temporary embolization is desired. Degradable hydroxyethyl acrylate (HEA) microspheres have been synthesized. Canine renal arteries and rabbit central auricular arteries were embolized with HEA microspheres, and compared with degradable human serum albumin (HSA) microspheres, and permanent microspheres. HSA and HEA microspheres both achieved temporary occlusions. HSA and HEA microspheres were recanalizated at 1 and 3 weeks, respectively, while arteries occluded with permanent microspheres did not recanalize. All embolic microspheres led to tissue infarction, with the short-term HSA microspheres providing the least damage, and the permanent microspheres leading to extensive damage. Advantages of temporary embolization were not convincingly demonstrated since temporary occlusions still led to tissue infarction. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyethyl acrylate; Temporary; Occlusion; Microspheres; Hydrogel
1. Introduction Therapeutic embolization is the selective transcatheter blockage of blood vessels or diseased vascular structures. Ideally, the normal blood supply to surrounding tissues is simultaneously preserved. For example, uterine fibroid embolization (UFE) may be an alternative to hysterectomy for symptomatic fibroids [1,2]. UFE is the process of occluding the blood supply to benign uterine smooth muscle tumors to reduce their size and alleviate associated symptoms, including bleeding and pain. Current embolization materials in clinical use include metal coils, glue, and particles [3–8]. The majority of these agents are permanent. However, there are numerous clinical situations, e.g. trauma, postpartum hemorrhage, or gastrointestinal bleeding, in which temporary embolization is desired. The typical aim of temporary embolization is to stop blood flow, allowing hemostasis and healing of the injured blood vessel. As a temporary
*Corresponding author. E-mail address: [email protected] (A. Metcalfe). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.12.022
embolization agent degrades, the blood vessel may recanalize, reestablishing the normal blood supply. Gelatin sponges (Gelfoams) and autologous materials, e.g. fat, dura mater, muscle and autologous clot, have been used for temporary embolization [9,10]. The main advantage of these materials is their low cost and their inherent biocompatibility. The in vivo degradation of autologous materials, as well as gelatin sponges, relies on enzymatic action, that may vary widely between individuals and might not be predicted accurately [11,12]. Hydrolytically degradable materials could provide a mean to control the in vivo lifetime of an artificial embolus. Important additional properties would be resiliency, needed to pass through an orifice with a diameter smaller than the diameter of the microspheres and thus enabling highly selective embolization, and an industrially meaningful shelf life prior to use. Crosslinked hydrogel microspheres would fulfill the resiliency requirement. Moreover, through bulk degradation, their degradation times might be controllable through a combination of degradation pH (blood pH and storage pH), the number and type of crosslinks, and the monomers used.
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This initial study assessed the occlusion and recanalization that can be obtained following temporary arterial embolization with degradable hydrogel microspheres utilizing a novel hydroxylamine containing crosslinker.
2. Materials and methods 2.1. Materials All chemicals used were purchased from Aldrich (Milwaukee, WI) and were used as received. Methacryloyl chloride (Aldrich) was purified by vacuum distillation. 2.2. Synthesis of the crosslinker 2.2.1. Preparation of adipoyl dihydroxamic acid In a 1000 ml flask containing a magnetic stirring bar, methanol (500 ml) and dimethyl adipate (143 g, 0.813 mol) were sequentially added. The reaction was initiated by introducing an aqueous solution of hydroxylamine (124 g of 50 wt% aqueous solution, 1.87 mol) into the above system. After the reaction proceeded for 48 h at room temperature, the mixture was concentrated using a Rotavapors (Buchi Analytical Inc., New Castle, DE) and the product precipitated gradually. Adipoyl dihydroxamic acid was washed with cold water (4 C) three times. After vacuum drying at 45 C for 72 h, a white solid was obtained with a yield of 88% based on the feed amount of dimethyl adipate, and its 1H NMR spectrum was consistent with the molecular structure of adipoyl dihydroxamic acid. 2.2.2. Synthesis of N,N0 -(dimethacryloyloxy)adipamide N,N0 -(dimethacryloyloxy)adipamide (C6NCL) was prepared by reacting adipoyl dihydroxamic with methacryloyl chloride in well-dried glassware under nitrogen. Adipoyl dihydroxamic (17.6 g, 0.1 mol) and a mixture of dimethylformamide (DMF) (150 ml) and pyridine (40 ml) were charged first. Methacryloyl chloride (22 ml, 0.21 mol) was dissolved in 20 ml DMF and this solution was added dropwise within 35 min with stirring. After reacting for 2 h at 35 C, 200 ml of chloroform was added to dilute the reaction mixture. Then, 15 ml of concentrated hydrochloric acid was diluted with 200 ml of water and this solution was transferred into the above reaction system. The water phase was extracted once with 50 ml of chloroform and the extract was combined with the organic phase. The chloroform solution was washed with water three times and dried with magnesium sulfate overnight. When concentrated by evaporating, C6NCL precipitated as white crystals, which were washed with ethyl ether twice and vacuum-dried over-
night at 35 C (yield: 38%). The molecular structure and high purity (>99%) were confirmed by 1H NMR. 2.3. Preparation of polymeric hydrogel microspheres All hydrogel microspheres were synthesized in the following manner as exemplified for HEA microspheres. A 500 ml open-mouth jacketed flask was equipped with a mixer, a thermometer and a temperature controller, and 150 ml of mineral oil and 0.12 g of sorbitan sesquioleate (SSO) were sequentially added. This system was heated to 60 C, while the water phase was prepared in a small beaker as follows. Sodium chloride (23.2 g) and sodium acetate (11.0 g) were first dissolved in distilled water (81.6 ml). Then, this aqueous solution was mixed with glycerol (163 ml) with magnetic stirring. Finally, the pH value of this mixture was regulated to 6.0 by adding acetic acid. The buffer solution (pH=6, 26 ml) was used to dissolve hydroxyethyl acrylate (5.0 g) and the color monomer 5fluorescein acrylate (10 mg). To this solution, the crosslinker C6NCL (0.3 g in 3.0 g DMF solution) was added dropwise with stirring. The mixture was heated to 60 C in an oil bath. As soon as the initiator ammonium persulfate (0.2 g) was added, this water phase was transferred into the oil phase with fast stirring (650 rpm), and tetramethylethylenediamine (0.4 ml) was added immediately to accelerate the reaction. The polymerization proceeded for 1 h; the mixture was rinsed into 120 ml of water to separate the microspheres from the oil. The orange microspheres were washed with water at least five times, and stored in a buffer (pH=2) in the refrigerator (4 C). 2.4. Human Serum Albumin microspheres Human Serum Albumin (20 g) was dissolved in 75 ml of HEPES buffered solution. The final volume was approx. 100 ml. To this solution was added 10 g of 1ethyl-3(3-dimethylaminopropyl) carbodiimide, quickly stirred, and immediately poured into 800 ml of mineral oil with 0.8 g of SSO, with overhead stirring at room temperature for 16 h. The emulsion was poured into 300 ml water and the oil phase was decanted. The microspheres were further purified by repeated centrifugation with water. The microspheres were sieved to 300–500 mm specification and stored in sterile water until use. 2.5. In vitro degradation studies Screening experiments were conducted for degradation and stability of the various homopolymer microspheres synthesized. Approximately 2 ml of microspheres were placed into a glass vial and 10 ml 50 mm phosphate buffer at pH 7.4, or 10 ml 5 mm
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glycine buffer at pH 2, were added. The microspheres were allowed to settle and the height of the settled microspheres was marked. The microspheres in physiological buffer (pH 7.4) were then placed into a 37 C oven and the microspheres in storage buffer (pH 2.0) were placed into the refrigerator at 4 C. The vials were shaken once a day and the volume of settled microspheres was noted. The microspheres were deemed to have degraded after no microspheres were detectable by visual means.
2.6. In vivo studies The HEA microspheres were synthesized as described above and sieved to obtain 300–500 mm microspheres. The microspheres had a degradation time of approximately 3 weeks in vitro. The microspheres were stored in pH 2 glycine buffer; immediately prior to embolization, the storage buffer was repeatedly replaced with phosphate buffered saline, at physiological pH, until the solution reached neutral pH. As control for permanent embolization, EmboGolds microspheres (Biosphere Medical, Inc., Rockland, MA) of 300–500 mm size were utilized. As a control for temporary occlusion of the polymer microspheres, HSA microspheres 300–500 mm size were used. Protocols for animal experimentation were approved by the institutional Animal Committee in accordance with guidelines of the Canadian Council on Animal Care.
2.6.1. Renal arteries occlusion model Five beagles weighing 10–15 kg were sedated with an intramuscular injection of acepromazine (0.1 mg/kg), glycopyrrolate (0.01 mg/kg), and butorphanol (0.1 mg/ kg) and were anesthetized with intravenously administered thiopental (15 mg/kg). The animals were ventilated artificially and maintained under surgical anesthesia with 2% isoflurane. A right percutaneous femoral artery puncture was followed by selective catheterization of renal arteries using 4 or 5 F catheters (Terumo, Thailand). Following a diagnostic angiogram to document the anatomy and distribution of the renal arteries, progressive embolization of the posterior division was performed under fluroscopic and angiographic guidance, attempting to occlude 50% of the supply to the renal parenchyma, and taking care to avoid reflux into the other division or outside the renal artery territory. A total of 10 kidneys in 5 animals were studied for 3 weeks by immediate and weekly catheter angiography, followed by macroscopic photography and pathology. Three renal arteries were embolized with HEA microspheres, 3 with permanent particles, 2 with HSA microspheres, and 2 kidneys were not embolized.
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2.6.2. Median auricular artery occlusion model Five New Zealand rabbits weighing 2.5–3.5 kg were sedated with an intramuscular injection of acepromazine (0.75 mg/kg), and glycopyrrolate (0.01 mg/kg). Local preoperative analgesia was provided with application of Emla cream on ears. Catheterization of central artery of rabbit ears was performed with radiopaque catheters (Protectivs Plus, Johnson & Johnson Medical, Arlington, TX). Following a diagnostic angiogram to document the anatomy and distribution of the auricular arteries, progressive embolization of the central auricular artery with embolic microspheres was performed under fluroscopic and angiographic guidance. Four central auricular arteries were embolized with HEA microspheres, 3 with permanent particles and 3 were not embolized. Preoperative and postoperative analgesia was provided by buprenorphine (0.05 mg/kg). Immediate angiography was undertaken via the same catheter used for injection. During recovery, the rabbits were fed a normal diet and their activities were not restricted. All ears were visually studied twice a week to assess any ear or artery changes. Rabbits (10 ears) were studied at 1 and 2 weeks by catheter angiography, followed by macroscopic photography and pathology.
3. Results 3.1. Characterization of polymer microspheres The in vitro stability and degradation was designed to mimic in vivo events by placing microspheres into a 37 C oven, and shelf life by placing the microspheres into the refrigerator. A variety of different acrylic monomers were homopolymerized with C6NCL and the degradation times found for the various polymeric microspheres are summarized in Table 1. 3.2. In vivo experiments All animals survived without adverse effects. When HEA microspheres were used for embolization, renal arterial occlusions that persisted at 1 week were recanalized at 3 weeks (Fig. 2). For comparison, renal arterial branches embolized with HSA microspheres recanalized at 1 week, while arteries embolized with permanent microspheres did not show recanalization at 3 weeks. On macroscopic inspection after sacrifice, small infarcts were visible in kidneys embolized with temporary microspheres, while much more extensive abnormalities were noted in those embolized with permanent microspheres, with a significant decrease in the overall size of the kidney (Fig. 3). Following sectioning and histopathological processing, all kidneys treated with permanent microspheres
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Table 1 Homopolymer microspheres evaluated and their respective degradation times Monomer
Crosslinkera
Degradation Storage timeb degradation timec
TS HEA HMMA PEG-macromer AA NaAA DMA AAm
C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL
17 days 22 days 20 days 31 days 8h 6h 32 h 7h
None detected None detected None detected None detected o1 week o1 week E1 week o1 week
Monomers used: N-[tris(hydroxymethyl)methyl]acrylamide (TS), 2hydroxyethyl acrylate (HEA), N-(hydroxymethyl)methacrylamide (HMMA), poly(ethylene glycol)-methacrylate (PEG-macromer, MW average 526), acrylic acid (AA), sodium acrylate (NaAA), N;Ndimethylacrylamide (DMA), and acrylamide (AAm). a Hydrogel formed with 5% crosslinker at 55 C. b pH=7.4, 37 C. c pH=2; 5 C; >2 months.
Fig. 1. Synthesis and degradation of HEA microspheres: (a) synthesis of crosslinker C6NCL and (b) degradation of crosslinked polymers using C6NCL crosslinker.
Fig. 2. Angiographic demonstration of transient occlusion. Right renal angiogram demonstrates renal arterial branch occlusion, with HEA microspheres, immediately after embolization (arrows in a), but that recanalized at 3 weeks (b). A small cortical infarct is visible as a nephrographic contour defect at 3 weeks (box in b).
Fig. 3. Macroscopic findings after embolization. On macroscopic inspection after sacrifice, small infarcts were visible in kidneys embolized with temporary microspheres (center), while much more extensive abnormalities were noted in those embolized with permanent microspheres (right), with a significant decrease in the overall size of the kidney as compared to the intact kidney (left). A focal infarct is visible despite the use of temporary microspheres (arrows).
Fig. 4. Histopathological findings after embolization. Following sectioning and histopathological processing, kidneys treated with permanent microspheres showed extensive infarction; further, the embolic particles were clearly visible (arrows in a). The embolic spheres tended to be close to the cortical surface of the organs, where they were accompanied by a moderate inflammatory reaction. In kidneys embolized with degradable microspheres, cortical infarctions could also be found. Microspheres, sometimes intact but encapsulated, but most often in various stages of degradation and phagocytosis, could also be detected (arrows in b). In certain locations, only a residual inflammatory reaction and some neointimal thickening could be observed as a witness to the previous presence of these degradable microspheres (arrow in c). Normal renal parenchyma is shown in d for comparison. Movat’s pentachrome stain, original magnification: 20 (a), 50 (b, d), 100 (c).
showed extensive infarction (Fig. 3); further, the embolic particles were clearly visible. The embolic particles tended to be close to the cortical surface of the organs, where they were accompanied by a moderate inflammatory reaction. In the kidneys embolized with degradable microspheres, cortical infarctions could also be found. Microspheres, sometimes intact but encapsulated, but most often in various stages of degradation and phagocytosis, could also be detected (Fig. 4b). In certain
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locations, only a residual inflammatory reaction and some neointimal thickening could be observed as a witness to the previous presence of these degradable microspheres (Fig. 4c). Experiments performed in the rabbit central auricular arterial model showed that HEA microspheres led to occlusions that persisted at 1 week but that recanalized at 2 weeks, while embolization with permanent microspheres did not recanalize. Temporary microsphere embolization caused some inflammation around the artery, but no necrosis. Visual observation under transillumination did not reliably show microspheres that were still poorly visible despite the use of fluorescein acrylate. Histopathological findings after sacrifice did not differ from those found in canine specimens.
4. Discussion Temporary embolization is intended for hemorrhagic episodes; too rapid degradation might lead to rebleeding, while a more persistent agent may lead to permanent occlusion of the injured vessel. Recanalization of the clot formed after coil occlusion in canine models typically occurs between 1 and 2 weeks after embolization, while arteries still occluded at 3 weeks cannot recanalize [13]. This experimental result was the starting point of our investigation of an embolic agent with reliable in vivo degradation times of 2–3 weeks. This time frame is also similar to the theoretical degradation time of the common embolic agent gelatin sponge [11]. There are two archetypal ways to render a polymeric material hydrolytically degradable. The first way is to use hydrophobic linear polymers, such as poly(lactic acid/glycolic acid) (PLGA), polyanhydrides, polyesters, and polyesteramides, that degrade into soluble monomers and oligomers [14]. For example, PLGA microspheres have been utilized for embolization [15], but the minimum time for degradation is on the order of months. PLGA microspheres lack resiliency; they cannot deform and regain their shape when pushed through a smaller catheter than the diameter of the microspheres. A second way to design a temporary agent is the use of hydrolytically degradable crosslinked polymers. For example, hydrolytically degrading polymers were synthesized in situ using photopolymerization of monomers in the presence of crosslinkers containing a hydrolytically unstable lactic acid moiety [16]. The degradation time of these polymers was a function of the number of lactic acid moieties incorporated into the crosslinker and the final polymer [17]. The lactic acidcontaining crosslinker must be stored under anhydrous conditions due to its ready hydrolysis. Other cross-
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linkers have been prepared containing hydrolytically labile carbonate [18], ester [19] and phosphazene moieties [20]. Hydrogels comprising such crosslinked polymers are not stable under aqueous storage conditions and start to degrade immediately following placement in an aqueous environment at any pH value. They would thus have to be dried and rehydrated prior to the actual embolization procedure. A crosslinked polymeric material may also be rendered hydrolytically degradable by incorporating crosslinks which are stable under either basic or acidic storage conditions, but which degrade at physiological pH. Ruckenstein and Zhang [21] described a base-stable crosslinker containing hemiacetal functions, accounting for its stability at high pH, and instability under acidic conditions. The degradation products are the linear polymer chains, ethylene glycol and acetaldehyde; however, the latter two molecules are undesirable in vivo. Ulbrich et al. have described a crosslinker, N, Odimethacryloylhydroxylamine, which is stable at low pH, but which degrades at physiological pH [22,23]. The degradation of the Ulbrich crosslinker is postulated to occur via base-catalyzed Lossen rearrangement of substituted hydroxamic acids and leads to linear polymer chains and carbon dioxide. In first screening experiments for stability under physiological and storage conditions under refrigeration utilizing this crosslinker with a variety of monomers, two extremes were noticed. For charged monomers, e.g. acrylic acid, the polymers at 5% crosslink density degraded readily within a couple of hours; even under storage conditions, which were highly unfavorable to the Lossen rearrangement, the microspheres degraded rapidly. For hydroxy containing monomers, e.g. hydroxyethyl acrylate, the polymers with 5% crosslink density degraded within a couple of months. In order to reduce the degradation time for such polymers, lower amounts of crosslinker were used, but this in turn led to fragile microspheres that fragmented when pushed through a catheter. Mixing of the two different acrylic monomer groups produced intermediate degradation times, but the composition was highly sensitive to the relative concentration of monomers and this was deemed difficult to scale-up reliably. Homopolymer microspheres are much more desirable as the scale-up would be much easier. The Ulbrich crosslinker’s inability, in our hands, to balance the degradation time with the mechanical properties required of the microspheres led us to design a new crosslinker, N,N0 -(dimethacryloyloxy)adipamide. In analogy to research into lactic acid containing crosslinkers [17], introduction of two degradation sites into the crosslinker might lead to a decrease in degradation time, while maintaining the desired mechanical properties. Care has to be taken that the degradation products released are essentially non-toxic.
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The degradation of substituted hydroxylamines leads to primary amines and carboxylic acid with the loss of carbon dioxide. This is the case with C6NCL, whose degradation products are a biogenic and non-toxic amine, putrescine, and the linear polymer chains. The synthesis of the new crosslinker was straightforward (Fig. 1). In initial screening experiments at fixed crosslinker ratio of 5%, the same phenomena described for the Ulbrich crosslinker was found (Table 1). For charged monomers, e.g. acrylic acid, their polymers degraded readily within a couple of hours; even under storage conditions the microspheres readily degraded. For uncharged monomers, e.g. hydroxyethyl acrylate, the degradation of their polymers under physiological conditional was approximately 3 weeks and the microspheres under storage did not show any swelling even after 3 months, indicating an industrially meaningful storage period. The combination of hydroxyethyl acrylate with the crosslinker C6NCL was chosen for further studies as it combined the required degradation time and mechanical resiliency. However, one drawback of the degradable hydrogel microspheres was their complete transparency. In a darkened cathlab, they were practically invisible in a syringe and for practical reasons, the microspheres were colored using small amounts of fluorescein acrylate resulting in orange microspheres. The two types of degradable microspheres, the HSA microspheres and the HEA microspheres described here, achieved the stated goal of reversibly occluding the vessels. In the case of HEA microspheres, occlusion lasted for the critical period at risk for recanalization, typically 10–14 days [13]. The degradation of the microspheres was followed by angiography at regular time intervals. While recanalization was demonstrated with both degradable microspheres and no recanalization in the case of the permanent embolic, the angiograms did not reveal the full extent of tissue infarction seen at sacrifice. All types of embolic microspheres led to tissue infarction. The extent of infarction clearly depended on the length of occlusion, with the short-term HSA microspheres causing the least damage, the permanent microspheres leading to severe tissue loss, and the HEA microspheres to moderate infarctions (Fig. 3). In contrast to the in vitro results seen for the degradation, the temporary HEA microspheres were not completely degraded in vivo and remnants were found in various stages of degradation (Fig. 4). This result could be explained by the potential change in extracelluar pH during ischemic events from approximately 7.4 to about 6.8 [24]. As the degradation is strongly pH dependent, such a seemingly small change in pH would prolong the time of degradation and this was confirmed in the laboratory (data not shown).
Previous studies [10–12] using degradable particles for temporary embolization relied on angiographic analysis of the occluded area. Recanalization was demonstrated and considered to be successful in not harming tissue close to the occlusion site. Recanalization of embolized sites in the kidney was demonstrated angiographically in our study too. However, the theoretical advantages of temporary embolization were not convincingly demonstrated in our model since temporary renal branch occlusions for the time periods investigated still led to tissue infarction. Although temporary embolic agents like HSA microspheres and the described HEA microspheres led to less pronounced tissue damage than permanent spheres, users should not be falsely reassured by the transient nature of the occlusion, and a meticulous technique and thorough knowledge and understanding of the anatomy are necessary to minimize ischemic complications.
5. Conclusion Temporary arterial occlusions for 2 weeks can be achieved using HEA microspheres. The clinical advantages of using such a material were not clearly demonstrated in our animal model.
Acknowledgements We thank Mrs. Lubba Shitsel for expert help in the synthesis of the various polymer microspheres.
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Biomaterials 25 (2004) 5209–5215
Transcatheter embolization using degradable crosslinked hydrogels Alexander Schwarza, Hongmin Zhanga, Annick Metcalfeb,*, Igor Salazkinb, Jean Raymondb b
a Biosphere Medical, Inc., 1050 Hingham Street, Rockland, MA 02370, USA Interventional Neuroradiology Laboratory, CHUM Research Center, Notre-Dame Hospital, Mailloux Pavilion M-8206, 1560 Sherbrooke East, Montreal, Que., Canada H2L 4M1
Received 5 December 2003; accepted 8 December 2003
Abstract Therapeutic embolization is the selective transcatheter blockage of blood vessels or diseased vascular structures. The majority of current embolization materials in clinical use are permanent. There are clinical situations however, in which temporary embolization is desired. Degradable hydroxyethyl acrylate (HEA) microspheres have been synthesized. Canine renal arteries and rabbit central auricular arteries were embolized with HEA microspheres, and compared with degradable human serum albumin (HSA) microspheres, and permanent microspheres. HSA and HEA microspheres both achieved temporary occlusions. HSA and HEA microspheres were recanalizated at 1 and 3 weeks, respectively, while arteries occluded with permanent microspheres did not recanalize. All embolic microspheres led to tissue infarction, with the short-term HSA microspheres providing the least damage, and the permanent microspheres leading to extensive damage. Advantages of temporary embolization were not convincingly demonstrated since temporary occlusions still led to tissue infarction. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyethyl acrylate; Temporary; Occlusion; Microspheres; Hydrogel
1. Introduction Therapeutic embolization is the selective transcatheter blockage of blood vessels or diseased vascular structures. Ideally, the normal blood supply to surrounding tissues is simultaneously preserved. For example, uterine fibroid embolization (UFE) may be an alternative to hysterectomy for symptomatic fibroids [1,2]. UFE is the process of occluding the blood supply to benign uterine smooth muscle tumors to reduce their size and alleviate associated symptoms, including bleeding and pain. Current embolization materials in clinical use include metal coils, glue, and particles [3–8]. The majority of these agents are permanent. However, there are numerous clinical situations, e.g. trauma, postpartum hemorrhage, or gastrointestinal bleeding, in which temporary embolization is desired. The typical aim of temporary embolization is to stop blood flow, allowing hemostasis and healing of the injured blood vessel. As a temporary
*Corresponding author. E-mail address: [email protected] (A. Metcalfe). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.12.022
embolization agent degrades, the blood vessel may recanalize, reestablishing the normal blood supply. Gelatin sponges (Gelfoams) and autologous materials, e.g. fat, dura mater, muscle and autologous clot, have been used for temporary embolization [9,10]. The main advantage of these materials is their low cost and their inherent biocompatibility. The in vivo degradation of autologous materials, as well as gelatin sponges, relies on enzymatic action, that may vary widely between individuals and might not be predicted accurately [11,12]. Hydrolytically degradable materials could provide a mean to control the in vivo lifetime of an artificial embolus. Important additional properties would be resiliency, needed to pass through an orifice with a diameter smaller than the diameter of the microspheres and thus enabling highly selective embolization, and an industrially meaningful shelf life prior to use. Crosslinked hydrogel microspheres would fulfill the resiliency requirement. Moreover, through bulk degradation, their degradation times might be controllable through a combination of degradation pH (blood pH and storage pH), the number and type of crosslinks, and the monomers used.
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This initial study assessed the occlusion and recanalization that can be obtained following temporary arterial embolization with degradable hydrogel microspheres utilizing a novel hydroxylamine containing crosslinker.
2. Materials and methods 2.1. Materials All chemicals used were purchased from Aldrich (Milwaukee, WI) and were used as received. Methacryloyl chloride (Aldrich) was purified by vacuum distillation. 2.2. Synthesis of the crosslinker 2.2.1. Preparation of adipoyl dihydroxamic acid In a 1000 ml flask containing a magnetic stirring bar, methanol (500 ml) and dimethyl adipate (143 g, 0.813 mol) were sequentially added. The reaction was initiated by introducing an aqueous solution of hydroxylamine (124 g of 50 wt% aqueous solution, 1.87 mol) into the above system. After the reaction proceeded for 48 h at room temperature, the mixture was concentrated using a Rotavapors (Buchi Analytical Inc., New Castle, DE) and the product precipitated gradually. Adipoyl dihydroxamic acid was washed with cold water (4 C) three times. After vacuum drying at 45 C for 72 h, a white solid was obtained with a yield of 88% based on the feed amount of dimethyl adipate, and its 1H NMR spectrum was consistent with the molecular structure of adipoyl dihydroxamic acid. 2.2.2. Synthesis of N,N0 -(dimethacryloyloxy)adipamide N,N0 -(dimethacryloyloxy)adipamide (C6NCL) was prepared by reacting adipoyl dihydroxamic with methacryloyl chloride in well-dried glassware under nitrogen. Adipoyl dihydroxamic (17.6 g, 0.1 mol) and a mixture of dimethylformamide (DMF) (150 ml) and pyridine (40 ml) were charged first. Methacryloyl chloride (22 ml, 0.21 mol) was dissolved in 20 ml DMF and this solution was added dropwise within 35 min with stirring. After reacting for 2 h at 35 C, 200 ml of chloroform was added to dilute the reaction mixture. Then, 15 ml of concentrated hydrochloric acid was diluted with 200 ml of water and this solution was transferred into the above reaction system. The water phase was extracted once with 50 ml of chloroform and the extract was combined with the organic phase. The chloroform solution was washed with water three times and dried with magnesium sulfate overnight. When concentrated by evaporating, C6NCL precipitated as white crystals, which were washed with ethyl ether twice and vacuum-dried over-
night at 35 C (yield: 38%). The molecular structure and high purity (>99%) were confirmed by 1H NMR. 2.3. Preparation of polymeric hydrogel microspheres All hydrogel microspheres were synthesized in the following manner as exemplified for HEA microspheres. A 500 ml open-mouth jacketed flask was equipped with a mixer, a thermometer and a temperature controller, and 150 ml of mineral oil and 0.12 g of sorbitan sesquioleate (SSO) were sequentially added. This system was heated to 60 C, while the water phase was prepared in a small beaker as follows. Sodium chloride (23.2 g) and sodium acetate (11.0 g) were first dissolved in distilled water (81.6 ml). Then, this aqueous solution was mixed with glycerol (163 ml) with magnetic stirring. Finally, the pH value of this mixture was regulated to 6.0 by adding acetic acid. The buffer solution (pH=6, 26 ml) was used to dissolve hydroxyethyl acrylate (5.0 g) and the color monomer 5fluorescein acrylate (10 mg). To this solution, the crosslinker C6NCL (0.3 g in 3.0 g DMF solution) was added dropwise with stirring. The mixture was heated to 60 C in an oil bath. As soon as the initiator ammonium persulfate (0.2 g) was added, this water phase was transferred into the oil phase with fast stirring (650 rpm), and tetramethylethylenediamine (0.4 ml) was added immediately to accelerate the reaction. The polymerization proceeded for 1 h; the mixture was rinsed into 120 ml of water to separate the microspheres from the oil. The orange microspheres were washed with water at least five times, and stored in a buffer (pH=2) in the refrigerator (4 C). 2.4. Human Serum Albumin microspheres Human Serum Albumin (20 g) was dissolved in 75 ml of HEPES buffered solution. The final volume was approx. 100 ml. To this solution was added 10 g of 1ethyl-3(3-dimethylaminopropyl) carbodiimide, quickly stirred, and immediately poured into 800 ml of mineral oil with 0.8 g of SSO, with overhead stirring at room temperature for 16 h. The emulsion was poured into 300 ml water and the oil phase was decanted. The microspheres were further purified by repeated centrifugation with water. The microspheres were sieved to 300–500 mm specification and stored in sterile water until use. 2.5. In vitro degradation studies Screening experiments were conducted for degradation and stability of the various homopolymer microspheres synthesized. Approximately 2 ml of microspheres were placed into a glass vial and 10 ml 50 mm phosphate buffer at pH 7.4, or 10 ml 5 mm
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glycine buffer at pH 2, were added. The microspheres were allowed to settle and the height of the settled microspheres was marked. The microspheres in physiological buffer (pH 7.4) were then placed into a 37 C oven and the microspheres in storage buffer (pH 2.0) were placed into the refrigerator at 4 C. The vials were shaken once a day and the volume of settled microspheres was noted. The microspheres were deemed to have degraded after no microspheres were detectable by visual means.
2.6. In vivo studies The HEA microspheres were synthesized as described above and sieved to obtain 300–500 mm microspheres. The microspheres had a degradation time of approximately 3 weeks in vitro. The microspheres were stored in pH 2 glycine buffer; immediately prior to embolization, the storage buffer was repeatedly replaced with phosphate buffered saline, at physiological pH, until the solution reached neutral pH. As control for permanent embolization, EmboGolds microspheres (Biosphere Medical, Inc., Rockland, MA) of 300–500 mm size were utilized. As a control for temporary occlusion of the polymer microspheres, HSA microspheres 300–500 mm size were used. Protocols for animal experimentation were approved by the institutional Animal Committee in accordance with guidelines of the Canadian Council on Animal Care.
2.6.1. Renal arteries occlusion model Five beagles weighing 10–15 kg were sedated with an intramuscular injection of acepromazine (0.1 mg/kg), glycopyrrolate (0.01 mg/kg), and butorphanol (0.1 mg/ kg) and were anesthetized with intravenously administered thiopental (15 mg/kg). The animals were ventilated artificially and maintained under surgical anesthesia with 2% isoflurane. A right percutaneous femoral artery puncture was followed by selective catheterization of renal arteries using 4 or 5 F catheters (Terumo, Thailand). Following a diagnostic angiogram to document the anatomy and distribution of the renal arteries, progressive embolization of the posterior division was performed under fluroscopic and angiographic guidance, attempting to occlude 50% of the supply to the renal parenchyma, and taking care to avoid reflux into the other division or outside the renal artery territory. A total of 10 kidneys in 5 animals were studied for 3 weeks by immediate and weekly catheter angiography, followed by macroscopic photography and pathology. Three renal arteries were embolized with HEA microspheres, 3 with permanent particles, 2 with HSA microspheres, and 2 kidneys were not embolized.
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2.6.2. Median auricular artery occlusion model Five New Zealand rabbits weighing 2.5–3.5 kg were sedated with an intramuscular injection of acepromazine (0.75 mg/kg), and glycopyrrolate (0.01 mg/kg). Local preoperative analgesia was provided with application of Emla cream on ears. Catheterization of central artery of rabbit ears was performed with radiopaque catheters (Protectivs Plus, Johnson & Johnson Medical, Arlington, TX). Following a diagnostic angiogram to document the anatomy and distribution of the auricular arteries, progressive embolization of the central auricular artery with embolic microspheres was performed under fluroscopic and angiographic guidance. Four central auricular arteries were embolized with HEA microspheres, 3 with permanent particles and 3 were not embolized. Preoperative and postoperative analgesia was provided by buprenorphine (0.05 mg/kg). Immediate angiography was undertaken via the same catheter used for injection. During recovery, the rabbits were fed a normal diet and their activities were not restricted. All ears were visually studied twice a week to assess any ear or artery changes. Rabbits (10 ears) were studied at 1 and 2 weeks by catheter angiography, followed by macroscopic photography and pathology.
3. Results 3.1. Characterization of polymer microspheres The in vitro stability and degradation was designed to mimic in vivo events by placing microspheres into a 37 C oven, and shelf life by placing the microspheres into the refrigerator. A variety of different acrylic monomers were homopolymerized with C6NCL and the degradation times found for the various polymeric microspheres are summarized in Table 1. 3.2. In vivo experiments All animals survived without adverse effects. When HEA microspheres were used for embolization, renal arterial occlusions that persisted at 1 week were recanalized at 3 weeks (Fig. 2). For comparison, renal arterial branches embolized with HSA microspheres recanalized at 1 week, while arteries embolized with permanent microspheres did not show recanalization at 3 weeks. On macroscopic inspection after sacrifice, small infarcts were visible in kidneys embolized with temporary microspheres, while much more extensive abnormalities were noted in those embolized with permanent microspheres, with a significant decrease in the overall size of the kidney (Fig. 3). Following sectioning and histopathological processing, all kidneys treated with permanent microspheres
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Table 1 Homopolymer microspheres evaluated and their respective degradation times Monomer
Crosslinkera
Degradation Storage timeb degradation timec
TS HEA HMMA PEG-macromer AA NaAA DMA AAm
C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL C6NCL
17 days 22 days 20 days 31 days 8h 6h 32 h 7h
None detected None detected None detected None detected o1 week o1 week E1 week o1 week
Monomers used: N-[tris(hydroxymethyl)methyl]acrylamide (TS), 2hydroxyethyl acrylate (HEA), N-(hydroxymethyl)methacrylamide (HMMA), poly(ethylene glycol)-methacrylate (PEG-macromer, MW average 526), acrylic acid (AA), sodium acrylate (NaAA), N;Ndimethylacrylamide (DMA), and acrylamide (AAm). a Hydrogel formed with 5% crosslinker at 55 C. b pH=7.4, 37 C. c pH=2; 5 C; >2 months.
Fig. 1. Synthesis and degradation of HEA microspheres: (a) synthesis of crosslinker C6NCL and (b) degradation of crosslinked polymers using C6NCL crosslinker.
Fig. 2. Angiographic demonstration of transient occlusion. Right renal angiogram demonstrates renal arterial branch occlusion, with HEA microspheres, immediately after embolization (arrows in a), but that recanalized at 3 weeks (b). A small cortical infarct is visible as a nephrographic contour defect at 3 weeks (box in b).
Fig. 3. Macroscopic findings after embolization. On macroscopic inspection after sacrifice, small infarcts were visible in kidneys embolized with temporary microspheres (center), while much more extensive abnormalities were noted in those embolized with permanent microspheres (right), with a significant decrease in the overall size of the kidney as compared to the intact kidney (left). A focal infarct is visible despite the use of temporary microspheres (arrows).
Fig. 4. Histopathological findings after embolization. Following sectioning and histopathological processing, kidneys treated with permanent microspheres showed extensive infarction; further, the embolic particles were clearly visible (arrows in a). The embolic spheres tended to be close to the cortical surface of the organs, where they were accompanied by a moderate inflammatory reaction. In kidneys embolized with degradable microspheres, cortical infarctions could also be found. Microspheres, sometimes intact but encapsulated, but most often in various stages of degradation and phagocytosis, could also be detected (arrows in b). In certain locations, only a residual inflammatory reaction and some neointimal thickening could be observed as a witness to the previous presence of these degradable microspheres (arrow in c). Normal renal parenchyma is shown in d for comparison. Movat’s pentachrome stain, original magnification: 20 (a), 50 (b, d), 100 (c).
showed extensive infarction (Fig. 3); further, the embolic particles were clearly visible. The embolic particles tended to be close to the cortical surface of the organs, where they were accompanied by a moderate inflammatory reaction. In the kidneys embolized with degradable microspheres, cortical infarctions could also be found. Microspheres, sometimes intact but encapsulated, but most often in various stages of degradation and phagocytosis, could also be detected (Fig. 4b). In certain
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locations, only a residual inflammatory reaction and some neointimal thickening could be observed as a witness to the previous presence of these degradable microspheres (Fig. 4c). Experiments performed in the rabbit central auricular arterial model showed that HEA microspheres led to occlusions that persisted at 1 week but that recanalized at 2 weeks, while embolization with permanent microspheres did not recanalize. Temporary microsphere embolization caused some inflammation around the artery, but no necrosis. Visual observation under transillumination did not reliably show microspheres that were still poorly visible despite the use of fluorescein acrylate. Histopathological findings after sacrifice did not differ from those found in canine specimens.
4. Discussion Temporary embolization is intended for hemorrhagic episodes; too rapid degradation might lead to rebleeding, while a more persistent agent may lead to permanent occlusion of the injured vessel. Recanalization of the clot formed after coil occlusion in canine models typically occurs between 1 and 2 weeks after embolization, while arteries still occluded at 3 weeks cannot recanalize [13]. This experimental result was the starting point of our investigation of an embolic agent with reliable in vivo degradation times of 2–3 weeks. This time frame is also similar to the theoretical degradation time of the common embolic agent gelatin sponge [11]. There are two archetypal ways to render a polymeric material hydrolytically degradable. The first way is to use hydrophobic linear polymers, such as poly(lactic acid/glycolic acid) (PLGA), polyanhydrides, polyesters, and polyesteramides, that degrade into soluble monomers and oligomers [14]. For example, PLGA microspheres have been utilized for embolization [15], but the minimum time for degradation is on the order of months. PLGA microspheres lack resiliency; they cannot deform and regain their shape when pushed through a smaller catheter than the diameter of the microspheres. A second way to design a temporary agent is the use of hydrolytically degradable crosslinked polymers. For example, hydrolytically degrading polymers were synthesized in situ using photopolymerization of monomers in the presence of crosslinkers containing a hydrolytically unstable lactic acid moiety [16]. The degradation time of these polymers was a function of the number of lactic acid moieties incorporated into the crosslinker and the final polymer [17]. The lactic acidcontaining crosslinker must be stored under anhydrous conditions due to its ready hydrolysis. Other cross-
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linkers have been prepared containing hydrolytically labile carbonate [18], ester [19] and phosphazene moieties [20]. Hydrogels comprising such crosslinked polymers are not stable under aqueous storage conditions and start to degrade immediately following placement in an aqueous environment at any pH value. They would thus have to be dried and rehydrated prior to the actual embolization procedure. A crosslinked polymeric material may also be rendered hydrolytically degradable by incorporating crosslinks which are stable under either basic or acidic storage conditions, but which degrade at physiological pH. Ruckenstein and Zhang [21] described a base-stable crosslinker containing hemiacetal functions, accounting for its stability at high pH, and instability under acidic conditions. The degradation products are the linear polymer chains, ethylene glycol and acetaldehyde; however, the latter two molecules are undesirable in vivo. Ulbrich et al. have described a crosslinker, N, Odimethacryloylhydroxylamine, which is stable at low pH, but which degrades at physiological pH [22,23]. The degradation of the Ulbrich crosslinker is postulated to occur via base-catalyzed Lossen rearrangement of substituted hydroxamic acids and leads to linear polymer chains and carbon dioxide. In first screening experiments for stability under physiological and storage conditions under refrigeration utilizing this crosslinker with a variety of monomers, two extremes were noticed. For charged monomers, e.g. acrylic acid, the polymers at 5% crosslink density degraded readily within a couple of hours; even under storage conditions, which were highly unfavorable to the Lossen rearrangement, the microspheres degraded rapidly. For hydroxy containing monomers, e.g. hydroxyethyl acrylate, the polymers with 5% crosslink density degraded within a couple of months. In order to reduce the degradation time for such polymers, lower amounts of crosslinker were used, but this in turn led to fragile microspheres that fragmented when pushed through a catheter. Mixing of the two different acrylic monomer groups produced intermediate degradation times, but the composition was highly sensitive to the relative concentration of monomers and this was deemed difficult to scale-up reliably. Homopolymer microspheres are much more desirable as the scale-up would be much easier. The Ulbrich crosslinker’s inability, in our hands, to balance the degradation time with the mechanical properties required of the microspheres led us to design a new crosslinker, N,N0 -(dimethacryloyloxy)adipamide. In analogy to research into lactic acid containing crosslinkers [17], introduction of two degradation sites into the crosslinker might lead to a decrease in degradation time, while maintaining the desired mechanical properties. Care has to be taken that the degradation products released are essentially non-toxic.
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The degradation of substituted hydroxylamines leads to primary amines and carboxylic acid with the loss of carbon dioxide. This is the case with C6NCL, whose degradation products are a biogenic and non-toxic amine, putrescine, and the linear polymer chains. The synthesis of the new crosslinker was straightforward (Fig. 1). In initial screening experiments at fixed crosslinker ratio of 5%, the same phenomena described for the Ulbrich crosslinker was found (Table 1). For charged monomers, e.g. acrylic acid, their polymers degraded readily within a couple of hours; even under storage conditions the microspheres readily degraded. For uncharged monomers, e.g. hydroxyethyl acrylate, the degradation of their polymers under physiological conditional was approximately 3 weeks and the microspheres under storage did not show any swelling even after 3 months, indicating an industrially meaningful storage period. The combination of hydroxyethyl acrylate with the crosslinker C6NCL was chosen for further studies as it combined the required degradation time and mechanical resiliency. However, one drawback of the degradable hydrogel microspheres was their complete transparency. In a darkened cathlab, they were practically invisible in a syringe and for practical reasons, the microspheres were colored using small amounts of fluorescein acrylate resulting in orange microspheres. The two types of degradable microspheres, the HSA microspheres and the HEA microspheres described here, achieved the stated goal of reversibly occluding the vessels. In the case of HEA microspheres, occlusion lasted for the critical period at risk for recanalization, typically 10–14 days [13]. The degradation of the microspheres was followed by angiography at regular time intervals. While recanalization was demonstrated with both degradable microspheres and no recanalization in the case of the permanent embolic, the angiograms did not reveal the full extent of tissue infarction seen at sacrifice. All types of embolic microspheres led to tissue infarction. The extent of infarction clearly depended on the length of occlusion, with the short-term HSA microspheres causing the least damage, the permanent microspheres leading to severe tissue loss, and the HEA microspheres to moderate infarctions (Fig. 3). In contrast to the in vitro results seen for the degradation, the temporary HEA microspheres were not completely degraded in vivo and remnants were found in various stages of degradation (Fig. 4). This result could be explained by the potential change in extracelluar pH during ischemic events from approximately 7.4 to about 6.8 [24]. As the degradation is strongly pH dependent, such a seemingly small change in pH would prolong the time of degradation and this was confirmed in the laboratory (data not shown).
Previous studies [10–12] using degradable particles for temporary embolization relied on angiographic analysis of the occluded area. Recanalization was demonstrated and considered to be successful in not harming tissue close to the occlusion site. Recanalization of embolized sites in the kidney was demonstrated angiographically in our study too. However, the theoretical advantages of temporary embolization were not convincingly demonstrated in our model since temporary renal branch occlusions for the time periods investigated still led to tissue infarction. Although temporary embolic agents like HSA microspheres and the described HEA microspheres led to less pronounced tissue damage than permanent spheres, users should not be falsely reassured by the transient nature of the occlusion, and a meticulous technique and thorough knowledge and understanding of the anatomy are necessary to minimize ischemic complications.
5. Conclusion Temporary arterial occlusions for 2 weeks can be achieved using HEA microspheres. The clinical advantages of using such a material were not clearly demonstrated in our animal model.
Acknowledgements We thank Mrs. Lubba Shitsel for expert help in the synthesis of the various polymer microspheres.
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