Injectable chitosan hydrogels as doxycycline delivery system for abdominal aortic aneurysm treatment

摘要

Introduction: Sac embolization has potential to treat and prevent endoleak after endovascular aneurysm repair (EVAR), but presently commercialized embolizing materials have several drawbacks and lead to frequent recurrence. Previously, our team showed that the endothelial lining, a thin layer of cells at interior surface of blood vessels, has an important role in endoleak persistence and recurrence. A promising strategy consists in developing a sclerosing occlusive agent to be injected by catheter in aneurysm to prevent endoleaks. The injected matrix should present strong mechanical properties after gelation, be biodegradable, induce endothelial denudation but then become biocompatible for allow cell invasion and tissue healing. To that purpose, an injectable radiopaque chitosan-based thermogel containing doxycycline (DOX) was developed. DOX presents sclerosing properties at high concentrations. In addition it is a well-known inhibitor of matrix metalloproteinases (MMPs) a family of zinc endopeptidases responsible for extracellular matrix degradation characterizing aneurysmal pathology. This project's hypothesis is based on two-stage release of the drug from the hydrogel: a fast burst release leading to sclerosing effect and a slow release of the remaining drug giving MMP inhibition properties. Here we report preliminary results of the characterization and optimization of chitosan-hydrogels prepared with three different gelation solutions combined with DOX and a radiopaque agent (lodixanol), in regards to rheological, occlusive and injectability properties, biocompatibility and drug release rate. Methods: CH-DOX and CH gels were prepared by mixing a) a solution of CH dissolved in acidic solution containing lodixanol (GE Healthcare, USA) and b) a solution of gelation agent (BGP0.4M, SHC075MPB04M, SHC075MPB08M) with and without c) a solution containing DOX (Sigma). To increase the stability of DOX in solution, an antioxidant and a stabilizer was added. The gelation kinetic at 37°C was studied by rheology on a Physica MCR301 (Anton Paar). Cytotoxicity was evaluated on L929 fibroblasts and HUVEC, while the injectability through catheter (0.53 mm diameter) and the efficiency to occlude blood flow were evaluated on custom-made in vitro bench tests. Drug release rate was evaluated using an USP apparatus Ⅰ (Distek Dissolution equipment) at 37 °C during 60 hours. Results: All formulations immediately formed gels at physiological pH and body temperature. Adding lodixanol and DOX decelerate gelation, but to some extent increasing PB to 0.08M was able to counteract this effect. This formulation was injectable and immediately blocked the flow up to the maximum pressure generated by the bench system (＞220 mmHg). CH-OOX gels showed a two-stage release, with a burst release within the first 6 hours, followed by slow and continuous release (Fig 1). In vitro cytotoxicity test showed decrease of HUVEC viability for DOX concentrations higher than 1 mg/ml, suggesting that those concentrations could led to a sclerosing effect in vivo. Conclusion: Although further tests are required to confirm the endothelial removal and MMP inhibition effect, CH-DOX gel appears as a promising candidate for aneurysm embolization.