Hydrogels by Stereo-complexation of Water-soluble Plla-peo-plla and Pdla-peo-pdla Triblock- Copolymers

s / Journal of Controlled Release 72 (2001) 225 –309 247 Introduction Biotechnology has evolved to the point that it is possible to prepare therapeutic genes for most diseases. The use of these new medicines is hampered by the lack of a suitable carrier system that helps the DNA in reaching the target cells and in the intracellular trafficking. Cationic polymers have been proposed as vectors for genetic material since they readily form polyelectrolyte complexes with DNA. A drawback of using basic polymers like poly(L-lactic acid) is that they are able to interact with erythrocytes when they are placed in contact with blood. The purpose of this work is to synthesise new polymers based on poly-a-L-amino acids that are able to condense and protect DNA, show low cyto and haemotoxicity and enhance transfection efficiency compared to naked DNA. Results and discussion Polymers based on poly-L-glutamic acid are prepared via aminolysis of poly-g-benzyl / trichloromethyl-L-glutamate. In this way polymers were prepared that feature in their side chain tertiary amines in combination with primary amines, imidazole groups, guanidine functions, dithiopyridine groups, maleimide groups or hydrazide functions, depending on the future purpose. The molecular weight of these polymers were controlled by the monomer to initiator ratio. The physico-chemical properties of the new polymers were determined with EtBr fluorescence, photon correlation spectroscopy and titration studies. The interaction with erythrocytes was assessed by monitoring haemolysis and haemagglutination. The MTT assay was used to get more information on the cytotoxicity of the polymers. Acknowledgements This work was supported by the Flemish institute for the promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Flanders (FWO), the Belgian Ministry of Scientific Programming, IUAP/PAI-IV/11 and the European Union’s Biotechnology Programme Contract Number 97 2334 with support from INCO Contract Number IC 20 CT 970005. HYDROGELS BY STEREO-COMPLEXATION OF WATER-SOLUBLE PLLA-PEO-PLLA AND PDLA-PEO-PDLA TRIBLOCKCOPOLYMERS D.W. Grijpma and J. Feijen Institute for Biomedical Technology (BMTI) and Department of Polymer Chemistry and Biomaterials, Faculty of Chemical Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands Introduction This work describes the formation of new hydrogels for use in biomedical applications by the stereo-complexation of triblock-copolymers of PEO and Lor D-lactide from solutions in water. Poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) are known to form stereo-complexes upon blending or mixing [1,2]. By analogy, similar stereo-complexes are formed upon mixing diand triblockcopolymers, if the stereo-regular lactide sequences are of sufficient length [3,4]. Water-soluble triblock-copolymers containing polylactide (PLA) blocks can be prepared by initiating lactide polymerizations with polyethylene glycol (PEG) [5]. PLA-PEO-PLA triblock-copolymers that are soluble in water and at the same time are able to form water-insoluble stereo-complexes, offer exciting new possibilities in tissue engineering and in the controlled delivery of sensitive drugs like proteins and peptides. Materials and Methods PLA-PEO-PLA triblock-copolymers were prepared in bulk at 1308C by initiating either L-lactide or D-lactide polymerizations with PEG [3]. Land D-lactide were also polymerised under the same conditions to yield the stereo-regular homopolymers. PEGs of different molecular weights were obtained from Merck (Germany), polymer grade Land D-lactide monomers from Purac Biochem (The Netherlands) and the catalyst stannous octoate from Sigma (USA). All materials were used without further purification. The absolute molecular weights of the synthesized (triblock-co)polymers were determined by GPC (Waters system) in CHCl at 258C, 3 calibrated with narrow polystyrene standards with viscometer(Viscotec H502) and refractive index (Waters 410) detection. The 1 triblock-copolymer composition and the lactide monomer conversion were characterized by H-NMR (Varian 300 NMR). The solubility of the synthesized triblock-copolymers in water (1 g per 20 ml) was tested at 228C. Polymer films of (triblock-co)polymers were prepared by casting 1:1 mixtures of the L-lactide and D-lactide (triblock-co)polymers in CHCl or water. The 1:1 mixtures of L-lactide and D-lactide triblock-copolymers in water (1 g per 20 ml) were also freeze-dried. After 3 removal of the solvent, the solubility in water was again evaluated. DSC (Perkin-Elmer DSC-7) was used to show the formation of stereo-complexes in the obtained materials. 248 Abstracts / Journal of Controlled Release 72 (2001) 225 –309 Fig. 1. Solubility in water of PLLA-PEO-PLLA triblock-copolymers. The lactide to PEO mol ratio is twice the length of the lactide blocks. Results and discussion In the absence of PEG, which acts as an initiator in the ring opening polymerization of lactide, very high molecular weight Land 3 D-lactide homopolymers with number averaged molecular weights in excess of 100310 could be synthesized. Therefore, in the PLA-PEG-PLA triblock-copolymer synthesis the molecular weights and the lengths of the lactide blocks could precisely be tuned by adjusting the PEG to lactide ratio in the feed. GPC and NMR confirmed the living character of the PEG-initiated ring opening polymerization of lactide. The phase diagram of PLLA-PEO-PLLA triblock-copolymers in water is presented in Fig. 1. As PLA is hydrophobic and PEO is hydrophilic, the ratio of the lengths of the lactideand PEO blocks will determine the solubility of the triblock-copolymer. As the lactide content in the triblock copolymer increases, its solubility in water decreases. PLLA-PEO-PLLA triblock-copolymers with longer lactide sequences are only soluble in water if the molecular weight of the PEO block is high enough. Stereo-complexation of the Land D-lactide segments can only occur provided the length of these sequences is adequate. The 3 water-soluble triblock-copolymers with the highest PEO-block molecular weight of 13310 were further investigated in their complexation behaviour. Table 1 shows the formation of stereo-complexes in solvent cast films, as observed by DSC. Films prepared mixed PLLA and PDLA solutions in chloroform show a large melting endotherm at temperatures much higher than the regular melting temperature of the lactide homopolymers, indicating stereo-complexation. Polylactides melt at temperatures between 180 and 1908C, while the stereo-complex shows a peak melting temperature at approximately 2358C. Films prepared from mixtures of triblock-copolymers in chloroform and in water, in which the lactide sequences are 13 or 15 lactide units long, also show an endotherm at high temperatures. The melting temperature and the heat of fusion of these stereo-complexes is significantly lower than that of the homopolymers. Nevertheless, even in films prepared from solutions in water, stereo-complexation can be observed. Once the films are formed and stereo-complexation has taken place, these films are not soluble in water anymore. Freeze-drying was also carried out to remove the water part from these triblock-copolymer solutions in water. The obtained porous structures, with a porosity of approximately 95%, were not very strong, but could nevertheless be handled easily. Table 1 Stereo-complexation of 1:1 mixtures of lactide homopolymers and triblock-copolymers Complex Solvent Melting range (8C) PLLA1PDLA CHCl 203–246 3 15L-PEO-15L115D-PEO-15D CHCl 191–242 3 13L-PEO-13L113D-PEO-13D CHCl 183–241 3 13L-PEO-13L113D-PEO-13D water 183–241 Abstracts / Journal of Controlled Release 72 (2001) 225 –309 249s / Journal of Controlled Release 72 (2001) 225 –309 249 It is therefore possible to prepare water-insoluble polymeric materials with water as the only solvent. Current research is aimed at optimizing these systems and investigating their suitability for the controlled delivery of proteins and peptides. Conclusions PLA-PEO-PLA triblock-copolymers, where the PLA part consists of either Lor D-lactide can form water-insoluble stereo-complexes from solutions in water. The use of these hydrogels offers new perspectives in cell encapsulation and in the preparation of controlled protein and peptide delivery systems. AcknowledgementsWe would like to acknowledge the assistance of Ir. Wendy D. van Dijk in the experimental work. References[1] G.L. Loomis, J.R. Murdoch, K.H. Gardner, Polym. Prepr. 31 (1990) 55.[2] H. Tsuji, S.H. Hyon, Y. Ikada, Macromolecules, 24 (1991) 5751.[3] W.M. Stevels, M.J.K. Ankone, P.J. Dijkstra, J. Feijen, Makromol. Chem. Phys., 196 (1995) 3687.[4] S.J. de Jong, W.N.E. van Dijk, J.J. Kettenes, P.J.W. Schuyl, W.E. Hennink, Macromolecules, 31 (1998) 6397.[5] I. Rashkov, N. Manolova, S.M. Li, J.L. Espartero, M. Vert, Macromolecules, 29 (1996) 50. NEW IONIC AMPHIPHILE BIOVECTORE AS CARRIER OF POOR SOLUBILITY DRUGSL. Imbertie, D. Betbeder, F. Lescure, P. Von Hoegen and I. De MiguelBiovectorTherapeutics, Chemin du Chene Vert, 31676 Labege Cedex, France, e-mail: [email protected] IntroductionImproving formulation capabilities and bioavailability of poorly soluble or lipidic drugs has been one of the most challenging fields inapplied pharmaceutical research. A wide variety of approaches has been developed including nanoparticles (1), lipid based colloidal systems(2), liposomes (3), solid lipid nanoparticles (4), lipobeads (5) and polymeric micelles (6). Depending on the route of administration anddrug characteristics, each system has its own advantages and limitations. Hydrophobic or amphiphilic systems allow to solubilise andstabilise non-soluble and hydrophobic compounds, but drug loading in the matrix is sometimes a difficult process and may lead to stabilityproblems. Limitations are generally related to drug loading, loading stability and stability of the particulate system in suspension. Surfactantsare often necessary to ensure the stability of the particulate system.Ion exchange gel matrices allow efficient loading of ionic compounds, but they lack stability under saline conditions. We thus deducedthat the combination of ionic and hydrophobic properties could be an interesting tool to obtain good solubilisation properties, stability ofdrug entrapment, stability of the particulate system as well as superior formulation properties.In this work such a new type of drug carrier combining ionic, hydrophilic and hydrophobic properties is described. The ionic amphiphilicproperties are obtained by inclusion of an ionic lipid phase into an oppositely charged ionic polymer hydrogel network. The resultingpolymer-lipid matrix is able to load easily and to stabilise a large spectrum of insoluble, hydrophobic or polar amphiphilic drugs due to thecombination of interactions. Charge sign and charge density, size (microor nanoparticles) or even degradability characteristics arepredefined by the chemical synthesis of the ionic hydrogel matrix. Ionicity and hydrophoby can be easily tailored afterwards by the quantityand type of lipid phase incorporated into the polymeric network.Entrapment and stability of Amphotericin B are presented as examples.