John R. Martin
Many different polymeric scaffold materials that exhibit degradability and minimal toxicity have been developed as bone void fillers to promote tissue regeneration in craniofacial bone defects, but in general these materials do not intrinsically promote new bone growth. To this end, implants coated with electrostatic layer-by-layer (LbL) assemblies of polyelectrolyte polymers and pro-healing growth factor proteins such as bone morphogenic protein-2 (BMP-2) have been developed. However, current polyelectrolyte-based constructs are engineered to degrade by non-specific hydrolysis to mediate protein release and are minimally-responsive to the rate of tissue repair. This limitation motivates the need for LbL systems that better deliver therapies over the entire lifetime of the craniofacial bone healing process. Therefore, we have developed stimuli-responsive LbL nanofilms that selectively release drug payloads in response to cell-generated reactive oxygen species (ROS), yielding a system that prolongs the drug efficacy window by conserving therapeutics only until they are needed. These LbL films employed a newly synthesized poly(thioketal-β-amino amide) (PTK-BAA) cationic polymer that is hydrolytically inert but degraded by ROS, thus mediating environmentally-responsive drug delivery. Methods: PTK-BAA polymers were synthesized by the 24hr condensation polymerization of two monomers, combining thioketal diamine diacryalamide with 4,4'-trimethylene dipiperidine in a solvent mixture of ethanol and water. LbL films were created on plasma treated poly(lactic-co-glycolic acid) (PLGA) scaffolds using tetralayers of PTK-BAA (+) / poly(acrylic acid) (PAA) (-) / BMP-2 (+) / PAA (-). Components were dissolved in 100mM acetate buffer at pH ≤ 5, and each successively dipped tetralayer was allowed to adsorb for 5-10min before being twice rinsed in an excess of deionized water. LbL films on PLGA scaffolds were incubated in PBS ± 1mM hydrogen peroxide (H2O2) at 37°C to evaluate protein release by ELISA. MC3T3-E1 pre-osteoblastic cells were treated with this LbL film releaseate for 48hrs before evaluating cellular alkaline phosphatase production at day 6 post-treatment. Finally, sterile PLGA scaffolds coated with LbL films comprising PTK-BAA and Alexa647-labled BMP-2 were implanted into 8mm rat calvarial defects and then excised at weeks 2 and 3 post-implantation to measure fluorescent signal remaining compared to day 0 levels using IVIS imaging. Results: ROS-degradable PTK-BAA polymers were synthesized and incorporated into LbL films with BMP-2 on PLGA scaffolds. The coatings experienced negligible BMP-2 release when incubated in PBS but discharged significantly greater protein amounts when treated with H2O2, indicating the ROS-specific functionality of these coatings. Furthermore, BMP-2 releaseate from H2O2-treated films increased in vitro markers of cellular osteogenesis compared with cells treated with PBS releaseate, indicating that ROS-mediated drug release kinetics correspond with protein bioactivity. Finally, PLGA scaffolds coated with PTK-BAA and fluorescently-labeled BMP-2 experienced sustained, multi-week protein release in rat calvarial defects, validating the use of ROS as a trigger for cell-mediated drug release in craniofacial applications. Conclusions: A new drug delivery platform that utilizes cell-generated ROS to mediate sustained delivery of therapeutic growth factor proteins has been developed. These data highlight the utility of PTK-BAA polymers as environmentally-responsive LbL film constituents that mediate selective payload release and corresponding drug bioactivity in vitro and in vivo, and emphasize the potential of “on-demand” drug delivery systems in craniofacial bone regenerative applications.