(633d) Tailoring Biomaterial Microcarriers for the Improved Delivery of Hemophiliac Factor IX

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) [1]. 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].