In vitro and in vivo protein delivery from in situ forming poly(ethylene glycol)–poly(lactide) hydrogels

https://doi.org/10.1016/j.jconrel.2007.03.014Get rights and content

Abstract

Previous studies have shown that stereocomplexed hydrogels are rapidly formed in situ by mixing aqueous solutions of eight-arm poly(ethylene glycol)-poly(L-lactide) and poly(ethylene glycol)–poly(D-lactide) star block copolymers (denoted as PEG–(PLLA)8 and PEG–(PDLA)8, respectively). In this study, in vitro and in vivo protein release from stereocomplexed hydrogels was investigated. These hydrogels were fully degradable under physiological conditions. Proteins could be easily loaded into the stereocomplexed hydrogels by mixing protein containing aqueous solutions of PEG–(PLLA)8 and PEG–(PDLA)8 copolymers. The release of the relatively small protein lysozyme (dh = 4.1 nm) followed first order kinetics and approximately 90% was released in 10 days. Bacteria lysis experiments showed that the released lysozyme had retained its activity. The relatively large protein IgG (dh = 10.7 nm) could be released from stereocomplexed hydrogels with nearly zero order kinetics, wherein up to 50% was released in 16 days. The in vitro release of the therapeutic protein rhIL-2 from stereocomplexed hydrogels also showed nearly zero order kinetics, wherein up to 45% was released in 7 days. The therapeutic efficacy of stereocomplexed hydrogels loaded with 1 × 106 IU of rhIL-2 was studied using SL2-lymphoma bearing DBA/2 mice. The PEG–(PLLA)8/PEG–(PDLA)8/rhIL-2 mixture could be easily injected intratumorally. The released rhIL-2 was therapeutically effective as the tumor size was reduced and the cure rate was 30%, whereas no therapeutic effect was achieved when no rhIL-2 was given. However, the cure rate of rhIL-2 loaded stereocomplexed hydrogels was lower, though not statistically significant, compared to that of a single injection with 1 × 106 IU of free rhIL-2 at the start of the therapy (cure rate = 70%). The therapeutic effect of rhIL-2 loaded stereocomplexed hydrogels was retarded for approximately 1–2 weeks compared to free rhIL-2, most likely due to a slow, constant release of rhIL-2 from the hydrogels.

Introduction

Recombinant human IL-2 (rhIL-2) is a broadly acting T cell-derived cytokine with proven anti-tumor activity, especially after local administration, and is produced by recombinant DNA technology [1]. Local IL-2 therapy is most effective against cancer when injected intratumorally [2]. In a clinical phase II trial, patients with advanced nasopharyngeal carcinoma were treated with combined radiotherapy and local rhIL-2 immunotherapy. The patients received 15 injections of rhIL-2 (3 times 5 daily injections in weeks 2, 4 and 6). After five years 63% of the patients were tumor-free, whereas treatment with only radiotherapy resulted in 8% tumor-free patients [3]. To avoid frequent and painful injections, a long acting protein delivery system is required.

Hydrogels have been used extensively as carriers for proteins, since their high water content renders them compatible with incorporated proteins and living tissue [4]. Injectable, in situ forming hydrogels are particularly interesting, because they allow easy and homogeneous loading of proteins [5]. Hydrogels can be formed by chemical and physical crosslinking. In situ forming physically crosslinked hydrogels have been prepared by a variety of noncovalent interactions, including self-assembly through hydrophobic interactions of poly(ethylene glycol) based block copolymers [6], [7], [8] or poly(N-isopropylacrylamide) (PNIPAAm) (co)polymers [9], [10], [11]. Crosslinking by physical interactions proceeds under milder conditions as compared to chemical crosslinking, which requires the use of photo-irradiation, organic solvents, auxiliary crosslinking agents and/or other reactive molecules that may damage the proteins to be incorporated. Recently, hydrogels have been prepared in situ from water-soluble poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) based block copolymers, in which the physical crosslinks are provided by stereocomplexation between the enantiomeric PLLA and PDLA blocks [12], [13], [14], [15], [16], [17], [18]. De Jong et al. have prepared stereocomplexed hydrogels from dextran–lactate graft copolymers [14] and Li et al. have prepared stereocomplexed hydrogels based on PLA–PEG–PLA triblock copolymers [15]. These stereocomplexed hydrogels have many advantages, e.g. they can be formed in situ at physiological conditions (37 °C, pH 7.4) by simply mixing two aqueous enantiomer solutions, the gelation process is very mild in which both temperature and pH do not change, and they are biodegradable. Nevertheless, dextran–PLA stereocomplexed hydrogels require involved synthesis of dextran–PLA graft copolymers, and stereocomplexed hydrogels based on PLA–PEG–PLA triblock copolymers exhibit relatively slow gelation and low mechanical strength.

The use of hydrogels for the release of rhIL-2 has been investigated [17], [18], [19]. Hanes et al. prepared rhIL-2 loaded microspheres by crosslinking of gelatin and chondroitin sulphate with gluteraldehyde. Release experiments in vivo using a brain tumor mice model showed a cure rate of 40% [19]. De Groot et al. prepared rhIL-2 loaded dextran-(hydroxyethyl)methacrylate (dex-(HE)MA) hydrogels by redox initiated polymerization [17]. When these hydrogels were used in vivo in SL2-lymphoma bearing DBA/2 mice, cure rates of 62% were obtained. Bos et al. studied release of rhIL-2 in vivo from stereocomplexed hydrogels based on dextran-L-lactate and dextran-D-lactate copolymers in this SL2-DBA/2 tumor mice model [18]. The therapeutic effect of rhIL-2 loaded hydrogels was at least comparable to injection of an equal dose with free rhIL-2 (cure rate of 60%).

We have previously reported on stereocomplexed hydrogels based on eight-arm PEG–PLA star block copolymers (PEG–(PLA)8) [13], [20]. The PEG–(PLA)8 copolymers could readily be prepared with controlled compositions. Upon mixing aqueous solutions of PEG–(PLLA)8 and PEG–(PDLA)8 copolymers, hydrogels with a high physical crosslinking density were rapidly formed. Rheological experiments showed that the hydrogel storage modulus increased with increasing PLA block length and polymer concentration, thus indicating a higher crosslinking density and a smaller hydrogel mesh size at higher PLA block length and higher polymer concentration. In this paper, the in vitro release of two model proteins with different hydrodynamic diameters, lysozyme and immunoglobulin G (IgG), were studied, as well as the release of the therapeutic protein rhIL-2. The therapeutic efficacy of rhIL-2 loaded stereocomplexed hydrogels was studied using the SL2 tumor mice model.

Section snippets

Materials

Eight-arm PEG–(PLLA)8 and PEG–(PDLA)8 star block copolymers were prepared as reported previously [13]. Lysozyme (from hen egg white) was purchased from Fluka (Buchs, Switzerland) and bovine immunoglobulin G (IgG, fraction II) was purchased from ICN Biochemicals BV (Zoetermeer, The Netherlands). Recombinant human interleukin-2 (rhIL-2) was purchased from Chiron BV (Amsterdam, The Netherlands). When the white lyophilized powder is reconstituted with 1.2 ml of water each vial contains per ml

Star PEG–PLA stereocomplexed hydrogels

Our previous studies showed that hydrogels were rapidly formed under physiological conditions upon mixing aqueous solutions of eight-arm poly(ethylene glycol)–poly(L-lactide) and eight-arm poly(ethylene glycol)–poly(D-lactide) star block copolymers (denoted as PEG–(PLLA)8 and PEG–(PDLA)8, respectively) via stereocomplexation of the PLLA and PDLA blocks [13], [20]. In this study, PEG–(PLLA)8 and PEG–(PDLA)8 star block copolymers (Mn, PEG = 21.8 kDa) with 12, 14 and 15 lactyl units per PLA block

Conclusions

Stereocomplexed PEG–PLA hydrogels were rapidly formed in situ by mixing aqueous solutions of PEG–(PLLA)8 and PEG–(PDLA)8 star block copolymers. These hydrogels degraded under physiological conditions and the single enantiomeric solutions had a low viscosity, thus allowing easy injection. Proteins could be easily loaded into the stereocomplexed hydrogels by mixing protein containing aqueous solutions of PEG–(PLLA)8 and PEG–(PDLA)8 copolymers. The in vitro release of the relatively small protein

Acknowledgements

This work was funded by the Netherlands Organization for Scientific Research (NWO).

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    Current address: Biomaterials Research Center, College of Chemistry and Chemical Engineering, Suzhou University, Suzhou, 215123, China.

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