Development of Super-Lubricious Platelet-Rich Plasma Loaded Microgels for the Treatment of Knee Osteoarthritis

Introduction: Knee osteoarthritis (OA) is a debilitating disease that significantly alters the microenvironment of the knee, increasing inflammation and reducing lubrication. This increased friction leads to significant wear and tear of articular cartilage, causing excruciating pain. Cartilage degeneration and inflammation proceed until a complete knee replacement is necessitated. It is currently estimated that over 27 million people in the United States alone suffer from varying degrees of OA, totalling ~$185 billion in socioeconomic costs. Furthermore, it is anticipated that 1 in 2 people will develop OA by the age of 85, resulting in over 67 million cases by 2030. As such, innovative treatments are needed to slow the inflammation and articular cartilage degeneration that characterize OA and to potentially restore joint homeostasis. One such approach is the delivery of platelet-rich plasma (PRP). PRP has shown mixed efficacy in delaying OA progression by releasing over 300 bioactive molecules that are critical to tissue regeneration and cellular recruitment. However, PRP delivery is hampered by rapid clearance from the synovial cavity. As such, we propose the use of polyethylene glycol (PEG) microgels to serve as delivery depots to prolong the half-life of PRP within the target area. PEG offers tunable swelling, mechanical, and degradation properties, allowing for intentional vehicle design while maintaining biomolecule bioactivity. Furthermore, our PEG microgels are coated with a custom-synthesized copolymer of dopamine methacrylate and sulfobetaine methacrylate poly(DMA-co-SBMA), offering a layer of polymer brush coating to improve the lubricity of the synovial fluid in the OA knee.

Materials and

Methods: PEG microgels (~100 µm) were fabricated using a modified electrospraying setup, where gelation was induced following droplet formation through the addition of triethanolamine to the oil bath containing the PEG droplets. This setup allowed for longer run times than traditional methods, where gelation occurred within the syringe after 10 minutes. Following washing and buffer exchange into phosphate buffered saline (PBS), PEG microgels were dip-coated in 10 mg/mL poly(DMA-co-SBMA) copolymer for 30 min. This copolymer was synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization and included batches in which rhodamine B acrylate was incorporated into the copolymer structure, allowing for easy visualization and detection. Spectroscopy and confocal microscopy confirmed successful adsorption of copolymer onto PEG microgels, while phase contrast microscopy was used to measure microgel size. To evaluate the mechanical properties of the microgels, a custom tribo-rheology setup was developed to form a ball-on-three-ball setup (Figure 1C), allowing for coefficient of friction measurements. Separately, injection force testing was performed to evaluate the injectability of these microgels. Microgels with and without copolymer coating were compared to PBS and simulated osteoarthritic synovial fluid. Coated PEG microgels were evaluated in vivo in a murine model for their stability and for pain. Microgels with encapsulated fluorescent particles were measured using an in vivo imaging system (IVIS) over 12 d and Von Frey and static weight-bearing tests were performed for mice injected with microgels or PBS sham. Lastly, PRP was encapsulated within PEG microgels and release followed via Bradford assay for 14 d.

Results, Conclusions, and Discussions: PEG microgels were successfully and consistently fabricated with diameter of ~100 µm using the modified electrospraying setup , though this diameter could be altered by changing electrospraying parameters (Figure 1A). Polydispersity of microgels following swelling in PBS was acceptable, with a coefficient of variance of ~20% (Figure 1B). Importantly, the modified electrospraying setup allowed for continuous operation for longer time periods, allowing for high amounts of microgels to be fabricated in one run. Custom synthesized poly(DMA-co-SBMA) copolymer showed nuclear magnetic resonance (NMR) and infrared peaks consistent with each of the monomers being incorporated into one cohesive structure. Furthermore, copolymer with integrated rhodamine B exhibited strong fluorescence that was correlated with copolymer concentration. PEG microgels swelled (20% increase in diameter) following buffer exchange into PBS but did not show significant change in size following coating with copolymer. Copolymer coating on PEG microgels persisted for at least 14 d. Importantly, coated microgels exhibited reduced coefficient of friction when compared to uncoated microgels and simulated OA synovial fluid (Figure 1C-D). Microgels were also shown to be easily injectable through 27 G needles. In vivo testing revealed coated PEG microgels were stable for at least 30 d and did not significantly change mice behaviour or pain threshold. Following encapsulation within PEG microgels, PRP exhibited prolonged release over 14 d (Figure 1E). The sustained release profile could partially be attributed to the formation of an interpenetrating clotting network formed within the PEG microgels, as evidenced by an increase in stiffness of PRP-loaded PEG microgels compared to PEG only samples. Work is currently underway to optimize copolymer structure by modifying the ratio of DMA to SBMA, as well as a block versus random copolymer arrangement. In vitro evaluation of super-lubricious PRP-loaded PEG microgels on chondrocytes in two-dimensional and three-dimensional cell culture is also underway.

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