Advanced Drug Delivery Reviews 54 (2002) 99���134 www.elsevier.com/locate/drugdeliv ABA-triblock copolymers from biodegradable polyester A- blocks and hydrophilic poly(ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins a , b *, Thomas Kissel Youxin Li , Florian Ungera a Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg, Ketzerbach 63, D-35032 Marburg, Germany b Schwarz Pharma AG, Monheim, Germany Received 3 July 2001 accepted 5 September 2001 Abstract Hydrogels are very attractive delivery systems for hydrophilic macromolecules such as proteins and DNA because they provide a protective environment and allow control of diffusion by adjusting cross-link densities. Physically cross-linked hydrogels generated by rapid swelling upon exposure to an aqueous environment can be obtained from ABA triblock copolymers containing hydrophobic polyester A-blocks and hydrophilic polyether B-blocks. They provide an attractive alternative to chemically cross-linked systems since they allow incorporation of macromolecular drug substances under mild process conditions. Moreover, they show controlled degradation behavior and excellent biocompatibility. In this review the synthesis and characterization of ABA triblock copolymers from polyester hard segments and poly(ethylene oxide) [PEO] soft segments as well as their biological and degradation properties will be discussed. Their use as biodegradable drug delivery devices in the form of implants, micro- and nanospheres has attracted considerable interest especially for proteins and may provide an alternative to poly(lactide-co-glycolide). ��� 2002 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable hydrogels ABA triblock copolymers Polyester/polyether block copolymers Synthesis Characterization Biodegradation Biocompatibility Protein delivery systems Abbreviations: LA, D,L-lactide LLA, L-lactide GA, Glycolide CL, e-Caprolactone PEO, Poly(ethylene oxide) or poly(ethylene glycol) PLA, Poly(D,L-lactide) PLLA, Poly(L-lactide) PGA, Poly(glycolide) PCL, Poly(e-caprolactone) PLGA, Random copolymer of D,L-lactide and glycolide PLC, Random copolymer of D,L-lactide and e-caprolactone PLLC, Random copolymer of L-lactide and e-caprolactone PLA���PEO���PLA, Triblock copolymer of poly(D,L-lactide-block-ethylene oxide-block-D,L-lactide) PLLA���PEO���PLLA, Triblock copolymer of poly(L-lactide-block-ethylene oxide-block-L-lactide) PLGA���PEO���PLGA, Triblock copolymer of poly[(D,L-lactide-co-glycolide)-block- ethylene oxide-block-(D,L-lactide-co-glycolide)] PLLGA���PEO���PLLGA, Triblock copolymer of poly[(L-lactide-co-glycolide)-block-ethyl- ene oxide-block-(L-lactide-co-glycolide)] PCL���PEO���PCL, Triblock copolymer of poly(e-caprolactone-block-ethylene oxide-block-e-cap- rolactone PLC���PEO���PLC, Triblock copolymer of poly(D,L-lactide-co-e-caprolactone-block-ethylene oxide-block-D,L-lactide-co-e-caprolac- tone) PLLC���PEO���PLLC, Triblock copolymer of poly(L-lactide-co-e-caprolactone-block-ethylene oxide-block-L-lactide-co-e-caprolactone) AB, Diblock copolymer of polyester���polyether ABA, Triblock copolymer of polyester���polyether���polyester BAB, Triblock copolymer of polyether���polyester���polyether DP, Degree of polymerization PDS, Parenteral depot system *Corresponding author. Tel.: 1 49-6421-282-5881 fax: 1 49-6421-282-7016. E-mail address: kissel@mailer.uni-marburg.de (T. Kissel). 0169-409X/02/$ ��� see front matter ��� 2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00244-7

100 T. Kissel et al. / Advanced Drug Delivery Reviews 54 (2002) 99 ���134 Contents 1. Synthesis and characterization of ABA triblock copolymers from poly(ethylene oxide) and biodegradable polyesters ..................... 100 1.1. Introduction ..................................................................................................................................................................... 100 1.2. Synthesis of block copolymers from PEO and lactones ....................................................................................................... 101 1.3. Mechanism of copolymerization from PEO and lactones..................................................................................................... 103 1.4. Physical properties of block copolymers from PEO and lactones ......................................................................................... 104 1.4.1. Microphase separation and crystallinity ................................................................................................................... 104 1.4.2. Hydrophilicity and swelling behavior ...................................................................................................................... 106 1.5. Conclusions ..................................................................................................................................................................... 107 2. Degradation and biological properties of ABA triblock copolymers from polyester A-blocks and PEO B-blocks............................ 107 2.1. Degradation of ABA triblock copolymers based on PEO and polyesters............................................................................... 107 2.2. Biocompatibility of ABA polymers ................................................................................................................................... 111 2.2.1. In vitro cytotoxicity/biocompatibility of ABA polymers ........................................................................................... 111 2.2.2. In vivo biocompatibility of ABA polymers .............................................................................................................. 113 3. Parenteral delivery systems based on ABA polymers ................................................................................................................. 116 3.1. Introduction ..................................................................................................................................................................... 116 3.2. Classification of parenteral depot systems .......................................................................................................................... 116 3.3. Overview on manufacturing techniques for PDS................................................................................................................. 117 3.4. Release mechanism of proteins and peptides from PLGA devices ........................................................................................ 118 3.5. Implants/devices from ABA polymers .............................................................................................................................. 119 3.6. Microspheres from ABA polymers as delivery systems for proteins ..................................................................................... 121 3.7. Nanoparticles from ABA polymers for drug targeting ......................................................................................................... 126 3.8. Release mechanism of proteins from ABA polymers .......................................................................................................... 128 3.9. Protein interactions with ABA polymers ............................................................................................................................ 128 4. Conclusions and perspectives ................................................................................................................................................... 129 References .................................................................................................................................................................................. 130 1. Synthesis and characterization of ABA reported in the literature. These different polymers triblock copolymers from poly(ethylene oxide) can be classified according to their structure as AB and biodegradable polyesters diblock, ABA or BAB triblock, multi-block, star- block and graft copolymers as shown in Fig. 1. 1.1. Introduction In this review we limit our discussions to polyes- ter/ether block copolymers of ABA and star-branch- Early attempts to use ���biomaterials��� which allow ed structures, where A designates a hydrophobic, replacement of diseased or defective limbs date back biodegradable polyester block, and B consists of to the ancient Egyptians and Greeks. Since then the PEO. The lack of a universally accepted nomencla- field has rapidly developed and with the advent of ture of ABA polymers makes a retrospective analysis synthetic materials numerous applications have been of literature data often difficult, if not impossible. identified [1]. Segmented block copolymers consist- Two systems have been proposed, namely the PELA ing of ���hard��� polyester A-blocks and ���soft��� poly- nomenclature giving the molecular weight (M ) of w (ethylene oxide) [PEO] B-blocks have attracted the the PEO block and the average degree of poly- interest of material scientists because they allow a merization of the A-blocks (e.g. PELA 6000/277) modification of physical and chemical properties, [3] and as a second approach adding the average leading to an accelerated biodegradability. These degree of polymerization as subscript to the block were considerations that stimulated research into new designation (e.g. PLA /PEO /PLA ) [4]. Since 109 41 109 block copolymers in the late 70s and block co- the data could often not be recalculated due to lack polymers of PEO and poly(terephthalate) for use in of information, we use the designation originally surgery were the first example for this new concept used in the literature. to our knowledge [2]. Since then numerous block or AB-diblock- and multiblock copolymers possess graft copolymers of PEO and various polyesters were micellar properties and have been studied as long-

T. Kissel et al. / Advanced Drug Delivery Reviews 54 (2002) 99 ���134 101 existing technologies for device manufacturing, which will be discussed later in this review. 1.2. Synthesis of block copolymers from PEO and lactones Homo- and copolymers of 6-hydroxycaprylic acid, lactic acid and glycolic acid are usually synthesized by the ring-opening polymerization of the cyclic monomers, e.g., e-caprolactone, lactide and gly- colide. In the last three decades, the block copoly- mers of poly(ethylene oxide) (PEO) and lactones have been investigated by many research groups. The terminal hydroxyl group of PEO can initiate successfully the polymerization of lactones in the presence of catalysts. Perret and coworkers were the first to our knowledge who prepared a series of block copolymers consisting of PEO and poly(e-caprolac- tone), PCL, by anionic polymerization in THF using naphthalene���sodium complex as catalyst [8]. The group of Cerrai and coworkers described block copolymers of PEO and PCL using a catalyst-free polymerization [9]. The polymerization of e-cap- rolactone with low M PEO was carried out in bulk w at 185 8C with almost quantitative conversion. They argued that the first step of the reaction involved the addition of two e-caprolactones to the terminal PEO hydroxyl groups yielding the corresponding bis-e- hydroxy diester, through selective acyl���oxygen bond Fig. 1. Schematic presentation of the architecture of block co- polymers from Ref. [103]. cleavage of the lactone ring, which then reacted further with e-caprolactone according to the usual ring opening polymerization mechanism. Therefore, circulating carriers for hydrophobic drugs after in- the copolymer formation proceeds, step by step, with travenous injection. Their potential as drug delivery the reaction between the hydroxyl function of the systems was reviewed recently [5,6]. regenerated e-hydroxycaproyl end unit and excess In almost all cases, the block copolymers were e-caprolactone. Polymerization of b-propiolactone synthesized using a variety of different catalysts, but and PEO was rather slow and did not lead to also catalyst free methods of synthesis have been quantitative conversion [10]. described. Synthesis of ABA-triblock copolymers of PEO The potential use of ABA triblock copolymers as B-blocks and lactic acid or glycolic acid A-blocks drug delivery system for hydrophilic macromolecular using Sb O and phosphoric acid as catalysts was 2 3 drugs, such as peptides and proteins was recognized first described by Cohn et al. in 1987 [3,11]. The in the early 90s [7]. ABA triblock copolymers can be copolymers were synthesized through the polycon- designed to exhibit rapid swelling upon contact with densation of lactic acid or glycolic acid in the water, forming a physically cross-linked, biodegra- presence of PEO under nitrogen flow. ABA polymer dable hydrogel. The advantage of this approach over compositions varied between 20 to 80 mol% poly- chemical cross-linking of PEO is the ease of incorpo- (lactic acid), PLA, with PEO chains in the 600���6000 ration of sensitive proteins and the broad spectrum of M range. They designated this new family of w

102 T. Kissel et al. / Advanced Drug Delivery Reviews 54 (2002) 99 ���134 biomaterials as PELA and proposed a nomenclature lactide in dichloromethane at 25 8C [29,30]. In the based on the M of PEO and the number average first step, the large 2,6-di-tert-butylphenoxy ligands w degree of polymerization of the PLA A-block, which are exchanged for the sterically less demanding was not universally accepted. They extensively in- alcohol ligands, then the alkoxide reacts with the vestigated mechanical and thermal behavior as well carbonyl group by formation of the ring opening as degradation and biocompatibility [12]. product. This catalyst system is very effective to Tin catalysts were frequently used in the ring obtain narrow M distribution product with high w opening polymerization of lactones [13���23]. Deng et conversion. al. used stannous chloride as catalyst to synthesize CaH and Zn metal as well as lithium chloride 2 successfully ABA triblock copolymers of PEO and were used as the heterogeneous catalyst system in PLA [13]. The polymerization was carried out in the copolymerization of PEO and lactones and bulk at 170���200 8C and yielded ABA polymers with usually are more suitable for synthesizing low Mw a single peak in GPC analysis and narrow polydis- block copolymers [31,32]. Catalyst-free polymeri- persity. Stannous octoate and metal oxides were used zations of lactones was always an ideal goal for by Kricheldorf et al. as the catalyst to prepare block polymers used as biomaterials. Cerrai et al. described copolymer of PEO and lactide [14]. With metal the synthesis of ABA triblock copolymers of L- oxides, such as GeO and SnO only low conver- lactide and PEO without catalyst [33]. Their results 2 2 sions of lactide were obtained, regardless of the demonstrated that their method is not competitive reaction temperature, whereas Sb O caused partial with catalyzed reactions due to a very long reaction 2 3 racemization of L-lactide and only SnO gave satisfac- time needed to get reasonable conversion of L-lac- tory results. However, with stannous octoate, racemi- tide. zation free ABA triblock copolymers were obtained Anionic polymerization was also used in the with a high conversion [14]. Similar results were preparation of the block copolymer of PEO and obtained by others [15,20,24]. Molecular weights of lactone. Jedlinski et al. synthesized block copolymer the ABA polymers corresponded to the feed ratios of of PEO and L-lactide through the anionic poly- monomer and initiator by an equation based on a merization of L-lactide in the presence of sodium simple chain reaction model. However, the GPC PEO alkoxide in THF at 25 8C [34]. The poly- trace showed a shoulder indicating that some homo- merization proceeds quickly and after 5 min the polymerization of lactide occurred. After fractiona- lactide was almost entirely consumed. The obtained tion, the shoulder peak was removed and a unimodal product exhibits a M higher than that of the w peak was obtained [15,20]. prepolymer and a unimodal M distribution. Selec- w Also aluminum triisopropoxide can be used as the tive extraction experiments showed that the com- catalyst in the copolymerization of L-lactide or L- position did not change, contrary to what is observed lactide/glycolide and PEO [25,26]. The polymeri- in a homopolymer blend. A slight racemization was zation was carried out in bulk at 150 8C and the observed during the polymerization. conversion was over 90%.The GPC trace showed a Kricheldorf and coworkers also reported the narrow M distribution and unimodal peaks. The anionic polymerization in the preparation of the w M s of the block copolymer are in agreement with block copolymer of PEO and lactide [35]. In their w the M calculated from the molar ratio of the experiments, they synthesized AB diblock and ABA w monomer and initiator as long as the M of the triblock copolymers of PEO���methyl ether or PEO w 1H-NMR product is not too high. also demonstrated and lactide in toluene at mild temperatures of 50 or 1H-NMR the block structure of the copolymers. Alkyl 80 8C using KOt���Bu as catalyst. Both and aluminum compound was also used in the copoly- GPC could prove the quantitative reaction of the merization of PEO and lactide [27,28]. PEOs with L-lactide. Recently, rare earth metal alkoxides were used for Star-shaped block copolymers of PEO and lactide the synthesis of ABA block copolymers in solution. were reported by several groups [28,36]. These Yttrium tris(2,6-di-tert-butylphenolate) was used by copolymers are prepared from multi-arm PEO, i.e. 4- Feijen et al. for the polymerization of PEO and or 8-arm PEO. Choi et al. prepared 2 -to 8-arm

T. Kissel et al. / Advanced Drug Delivery Reviews 54 (2002) 99 ���134 103 star-shaped block copolymers of PEO and L-lactide of 2-ethylhexanoic acid, does not seem to fit into this or e-caprolactone using stannous octoate as the scheme. Many investigations show that, most of the catalyst. Using triethylene aluminum as catalyst, we terminal groups of the product of the ring-opening synthesized 4- and 8-arm block copolymers with polymerization of lactones initiated by stannous L-lactide or L-lactide/glycolide. The polymerization octoate are hydroxyl groups, indicating that hydroxyl was carried out in toluene at 70 8C. GPC analysis groups, e.g., residual water or other impurities, showed an unimodal GPC trace and light scattering participate in the initiation as a co-catalyst. Macauley analysis showed a significant increase in M , corre- et al. studied the effect of hydroxyl and carboxylic w sponding to the ratio of monomer relative to multi- acid substances on the polymerization in the pres- arm PEO in the feed. Polydispersity was comparable ence of stannous octoate [16]. They found both, to that of the parent multi-arm PEOs. Connections of hydroxyl and carboxylic acid substances affecting ester blocks to ether blocks could be verified by the polymerization of lactide, but alcohol increased 1H-NMR. the PLLA polymerization rate and carboxylic acid decreased it. 1.3. Mechanism of copolymerization from PEO Du et al. reported their detailed investigation on and lactones the mechanism of the copolymerization of PEO and lactones in the presence of stannous octoate [23]. Although the mechanism of the polymerization of Kinetic measurement and mechanistic studies sug- lactone with many metal compounds remains some- gest that the reactivity of the initiator, a hydroxyl what speculative, the coordination-insert mechanism group-bearing reagent, is an important parameter on is most generally accepted, especially in the case of the polymerization mechanism. In the case of pri- metal alkoxides [14,15,20,26] (Fig. 2). mary and secondary alcohols, i.e. PEO and methyl In the presence of PEO molecules, the polymeri- lactate, it was found that when the initiator con- zation involves a transfer of metal alkoxide to metal centration exceeds the catalyst concentration, the PEOate [14,26]. The ring-opening polymerization is number of the propagation chains formed exceeded initiated by this macro-initiator of the metal alkoxide the number of catalyst molecules. The chains were as coordinated-insertion mechanism similar to low propagated through shifts of the catalysts from one M metal alkoxide catalysts. Stannous octoate, a salt chain to another. It was demonstrated that the w cooperation of stannous octoate with the terminated hydroxyl group of PEO formed a metal alkoxide 1H-NMR initiator. In situ was used to observe the mechanism of the copolymerization, PEO of Mw 1000 was used for the kinetic study (Fig. 3). Their results show the hydroxyl terminal group of PEO is esterified, and the number of the methylene protons were in agreement with those calculated from the M s of the final products, indicating that w the lactones are bound to the PEO chains. This evidence suggests that, both, stannous alkoxide and tin salts of carboxylic acids can initiate the ring opening polymerization, but the stannous alkoxide is more active than the salt. Thus, in the presence of alcohol, the initiation of ring opening polymerization of the lactones will be initiated by stannous alkoxide. In the case of aluminum alkoxide catalyst, i.e. aluminum triisopropoxide, the transfer of the aluminum ion on the terminal hydroxy group of PEO Fig. 2. Schematic representation of the coordination-insert mecha- nism for the polyesters. leads to the evaporation of isopropanol at high