Introduction: Current treatments for hemophilia B, a hereditary bleeding disorder characterized by the deficiency of clotting protein, factor IX, rely on injections and infusions that cause pain and discomfort, leading to noncompliance and risk of subsequent bleeding episodes. A non-invasive treatment using an oral delivery system can both overcome such issues and increase global access to protein therapy. Anionic complexation hydrogels have been engineered to protect therapeutic agents from the harsh environment of the GI tract and deliver them to the small intestine. We have successfully developed environmentally responsive biomaterial systems based on poly(methacrylic acid)-grafted-poly(ethylene glycol) [P(MAA-g-EG)] as delivery vehicles for factor IX (57 kDa) . We focused on optimizing P(MAA-g-EG)-based systems for oral delivery of human factor IX (hFIX) for hemophilia B treatment.
Methods: P(MAA-g-EG) films were synthesized with a pol(ethylene glycol) dimethacrylate crosslinking agent, varying in molecular weight (MW of PEG block=400-1000), and crosslinking densities (0.8-1.7 mol %) by UV-initiated free radical polymerization. Hydrogels were purified, dried, and crushed into 30-45 µm microparticles. Polymers were characterized by FTIR and potentiometric titration for polymer composition, SEM for size and morphology, and dynamic swelling studies for pH-responsive behavior. Cytotoxicity was assessed in Caco-2 cells using an MTS cell proliferation assay to screen for carriers that are potentially damaging to the small intestine. For protein loading, dried microparticles were swollen in an hFIX protein solution at pH 7.4 and remained in solution to allow for protein loading by diffusion. After loading was complete, microparticles were collapsed with acid to trap the protein in the polymer network, then rinsed to remove any surface loaded protein, and then lyophilized. Release studies were conducted following a two-stage dissolution procedure using simulated gastric fluid (SGF, pH 1.8) and fasted state simulated intestinal fluid (FaSSIF, pH 6.5). The amount of hFIX loaded and released was determined by an hFIX ELISA and protein activity was determined by a chromogenic assay. Permeability of hFIX was determined by transport studies with in vitro intestinal epithelial models.
Results: All formulation of P(MAA-g-EG) hydrogels consist of at least 96 mol % methacrylic acid. SEM images of crushed microparticles showed a wide polydispersity in size and irregular morphology. Hydrogels remained collapsed at pH ≤ 4 and then significantly swelled at above pH 5.2, which is the desired pH-responsive swelling for the transit through the GI tract. All formulation are cytocompatible with even high concentrations of 5 mg/mL showing no appreciable cytotoxicity. The degree of crosslinking affected the protein loading level, where decreased crosslinking density increased the loading level, reaching up to 60 µg hFIX/mg particle. Protein release in biorelevant media showed the desired release profile (Figure 1). Protein released in simulated intestinal conditions was at least 80% active.
Conclusions: Oral delivery of factor IX can be achieved by tailoring the biomaterial microcarriers to improve protein loading and release. Successful outcomes of this work will change hemophilia B treatment worldwide by offering a convenient and needle-free protein replacement therapy.
Acknowledgements: This work was supported in part by a grant from NIH (R01-EB-000246-20), an NSF-GRFP Fellowship and a P.E.O. Scholars Award to SDH, and the Fletcher S. Pratt Foundation. We also acknowledge the assistance of Joel Liou.
References: (1) Peppas NA and Horava SD. Polymers for Delivery of Therapeutic Proteins. U.S. Patent No. UTSB.P1047US.P1, Nov 2014. [Provisional application filed].