Co-cross-linked chitosan hydrogel as carrier for the local delivery of cisplatin. Liposome inclusion. Maria José Moura 1,2, Maria Helena Gil 2, Maria Margarida Figueiredo 2 1 Department of Chemical and Biological Engineering, Polytechnic Institute of Coimbra, 2 CIEPQPF, Department of Chemical Engineering, University of Coimbra, Results and Discussion Conclusions In vitro CDDP release studies Ionic cross-linked chitosan hydrogel Ionic/covalent co-cross-linked chitosan hydrogel Hydrogel micro-structure Fig. 1 presents the morphology and the pore size distribution of a chitosan hydrogel ionically cross-linked, and a matrix co-cross-linked with 0.10% of genipin, both imaged after water removal. An open network structure, highly porous, was observed for all the samples (> 90% porosity). The pores possess similar shapes, with a polygonal cross section, being those corresponding to the matrices co-cross-linked with genipin much larger and less uniform in size (200 µm vs 60 µm). FIG. 1 – Images of two hydrated samples (on the left) and corresponding SEM images and pore size distribution (MIP) of ionically cross-linked chitosan hydrogel (upper images) and genipin-co-cross-linked chitosan hydrogel (bottom images).  Hydrogels produced without and with genipin exhibit quite distinct morpho-structural properties.  T he interaction between chitosan and CDDP is more strong in the hydrogel ionically cross-linked. Fig. 2 and 3 presents the in vitro CDDP release from the chitosan hydrogel ionically cross-linked and from the matrix co-cross-linked with different genipin loads (0.10% and 0.20% genipin), respectively. CDDP loaded liposome, and then incorporated in both hydrogels, was also examined. FIG. 3 – In vitro cumulative percent release of CDDP from the co-cross-linked chitosan hydrogels with different genipin loads, in PBS at 37 ºC, measured by GFAAS. The initial CDDP loading is 0.6 mg/mL. FIG. 2 – In vitro cumulative percent release of CDDP from the chitosan hydrogel ionically cross-linked, in PBS at 37 ºC, measured by GFAAS. The initial CDDP loading is 0.6 mg/mL. All hydrogels exhibited rapid diffusion characteristics for the CDDP delivery reaching maximum release within 3 hours. The amount of drug released varied between 20% for the ionic hydrogel to about 60% for the co-cross-linked hydrogels. However, when the drug was encapsulated in liposomes the CDDP was completely released. Introduction Objectives Cisplatin [cis-diamminedichloroplatinum (II)], in short CDDP, is one of the most effective antitumor agents in the treatment of a variety of solid tumors such as ovarian, testicular, and bladder cancers [1]. This drug causes many severe toxic side effects, which frequently prevents the use of higher doses. A potential approach to overcome some of the drawbacks and optimize the oncologic treatment of this drug consists in developing carriers for chemotherapy able to release the drug directly in the tumor. Injectable in situ forming chitosan hydrogels are among the most successful models for such a purpose, since they are capable of forming stable gels at body temperature within a short period after injection. A novel in situ forming chitosan formulation has been recently developed, employing a reaction route where ionic and covalent co- cross-linking are combined [2]. The combination of chitosan with two cross-linkers, a glycerol-phosphate salt as an ionic cross-linker and genipin as a covalent cross-linker, leads to a thermosensitive hydrogel formed at mild conditions (pH≈7 and T≈37 ºC) with improved mechanical and chemical properties [2,3]. Experimental The aim of the present work is to assess the use of an ionic cross- linked chitosan hydrogel and an ionic/covalent co-cross-linked chitosan hydrogel for in situ release of CDDP. The use of a liposomal formulation, loaded with CDDP, and then incorporated in both hydrogels was also examined. 1.Casolaro M.; Cini R.; Del Bello B.; Ferrali M.; Maellaro E. Biomacromolecules: 10, (2009). 2.Moura M.J.; Faneca H.; Lima M.P.; Gil M.H.; Figueiredo M.M. Biomacromolecules: 12, (2011). 3.Moura M.J.; Figueiredo M.M.; Gil M.H. Biomacromolecules: 8, (2007). CDDP loaded liposome chitosan solution, 2% (in acetic acid, 0.5% v/v) pH ~ 5.5 glycerol-phosphate salt chitosan solution neutralized pH ~ 7 genipin (different loads) chitosan / genipin formulation pH ~ 7 co-cross-linked hydrogel curing (T=37 ºC; t=2h) CDDP in vitro drug release tests ionic cross-linked hydrogel curing (T=37 ºC; t=2h) or References Scanning Electron Microscopy (SEM) Mercury Intrusion Porosimetry (MIP) Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS) CDDP loaded hydrogels preparation Characterization methods  When CDDP was encapsulated in liposomes the interaction was negligible.