Poster P4 - Creation of dual-stage, ROS-responsive microparticles for critically-sized bone defects

Introduction: Regenerating large-scale bone defects in the clinic remains challenging. Specifically, segmental bone defects of greater than 2cm are unlikely to heal spontaneously, following skeletal stabilization alone and are thus deemed “critically” sized. Critically sized defects require planned reconstruction, with the gold standard biologic treatment being the iliac crest bone graft. However, there are many problems with this method: supply of autologous bone graft is limited and is associated with complications Furthermore, this harvesting requires an additional uncomfortable surgical procedure and has the additional disadvantages of quantity restrictions and substantial costs. To overcome these limitations with bone grafts, there has been considerable recent research efforts to regenerate bone endogenously by locally delivering osteogenic or vasculogenic growth factors to the defect site. Unfortunately, many of these tissue engineered constructs utilize traditional drug delivery systems which have limited drug payloads and feature passive, poorly controlled drug release kinetics. Additionally, most of these drug delivery systems only deliver a single osteogenic or vasculogenic therapeutic which poorly recreates processes seen in natural bone healing. These challenges motivate the development of new technologies that can controllably deliver both pro-angiogenic and pro-osteogenic therapies to promote complete restoration of large-scale bone defects.

We propose a two-compartment, environmentally-triggered drug delivery system with injectable microparticles. Previous studies have shown that reactive oxygen species (ROS) are key mediators of cell function, especially at sites of inflammation and tissue healing. This will allow for cell-mediated delivery of multiple therapeutics to areas high in ROS such as remodeling tissue.

Materials and

Methods: We propose to fabricate ROS-sensitive microparticles loaded with the osteogenic therapeutic Bone Morphogenic Protein-2 (BMP-2), and then coat the particles with ROS-sensitive layer-by-layer LbL films containing the pro-angiogenic therapeutic Vascular Endothelial Growth Factor (VEGF).

To investigate possible particle formulations, poly(propylene sulfide) (PPS), benzenethioketal (BTK), and polythioketal β-amino amide (PTK-BAA) were synthesized based on previous protocols, and poly(lactic-co-glycolic acid) (PLGA) was purchased from a vendor. Using either PPS, PLGA, BTK, or PPS & PLGA mixed, microparticles were formed via an oil/water/oil emulsion technique adapted from previous work. Microparticles were loaded with fluorescently tagged model protein, lysozyme (FITC-Lys). Particles were imaged via fluorescent microscopy to investigate drug distribution and particle stability. Scanning electron microscopy (SEM) and confocal microscopy were done to determine particles size and investigate agglomeration. Particles were dissolved in tetrahydrofuran (THF) to determine total loading and encapsulation efficiency. Additional particles will be incubated in various concentrations of hydrogen peroxide (H202) and evaluated for extended drug release.

LbL coatings were fabricated on stable microparticles via sequential incubation in charged solutions of PTK-BAA, a sacrificial anionic polymer, and model protein. Coatings were loaded with a different fluorescently tagged lysozyme (TRITC-Lys). Particle coatings were imaged via fluorescent microscopy to evaluate drug distribution. Coated particles were dissolved in THF and total loading and encapsulation efficiency of the LbL coating was examined. Particle formulations that were both loaded with FITC-Lys and coated with TRITC-Lys were imaged with fluorescent microscopy to show the dual payload capacity of the particles.

Results, Conclusions, and Discussions: Previous efforts have leveraged PPS in nanoparticle applications. However due to the nature in which particles are coated—successive incubations in aqueous solutions—difficulties quickly arose and it became clear that other polymers would need to be investigated.

Fluorescent microscopy in conjunction with SEM was used to assess particle agglomeration and stability. PPS particles lacked proper particle stability and would not form workable particles. While PLGA, PPS & PLGA, and BTK made stable particles, PLGA is not ROS-responsive. While the addition of PLGA to PPS helped to stabilize the particles, they still heavily agglomerated. Therefore our lead candidate formulation was chosen as the BTK particles, due to their combination of stability and oxidation-responsive behavior.

PPS, BTK, and PTK polymer structures were confirmed via nuclear magnetic resonance (NMR). Microparticles imaged using confocal microscopy and SEM were determined to be between 5-10 µm, small enough to still maintain injectability, while large enough to avoid phagocytosis. Particle loading was determined to be approximately 80 μg of drug per 20mg of particles. Additionally, preliminary work has shown dose dependent drug release based on peroxide concentration. This confirms the ROS responsiveness of our particle system and the extended drug release capabilities.

Fluorescent microscopy of coated particles confirmed LbL coating fabrication and even drug distribution. Loading was determined to be approximately 50 μg per layer, with a linear increase per layer. Additionally, fluorescent microscopy of combination core-loaded/coated particles, confirmed the system’s capacity for multiple therapeutics in discreet compartments.

Throughout this work we have evaluated multiple polymers for use in our dual-compartment coated microparticle system. While both PPS and PLGA have been extensively used in biomaterials research, their difficultly in forming stable particles means they are ultimately not useful for our application. BTK particles form highly stable particles that are sensitive to ROS oxidation. Therefore we have chosen the BTK polymer as our lead candidate and shown the formation of stable, ROS-sensitive, dual-stage microparticles is feasible. Future work will establish release kinetics, of single and multi drug payloads, and evaluate this system with therapeutic growth factors for in vivo bone regeneration.

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