Poster X8 - Post-synthesis modifications of poly(acrylic acid) microgels improves sustained IL-1Ra delivery for osteoarthritis treatment

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ruben Trujillo, MS

PhD Student
Cornell University
Ithaca, New York, United States

Co-Author(s)

SL

Sara Lok

Undergraduate Researcher
Cornell University, United States

Primary Investigator(s)

DP

David Putnam

Principal Investigator; Associate Dean for Innovation and Entrepreneurship
Cornell University, United States

Introduction: Osteoarthritis (OA) is a debilitating joint disease that affects millions of people across the world, leading to joint pain, inflammation, and a decreased quality of life. Treatment options for OA depend on the severity of the disease and include physical therapy, non-steroidal anti-inflammatory drugs, intra-articular (IA) injections of corticosteroids and viscosupplements, and ultimately total joint replacement. From a drug delivery perspective, IA injections are preferred, as they are a localized therapy, minimizing systemic side-effects. However, current IA injections of corticosteroids and viscosupplements do not slow the progression of OA, and there is a push towards finding disease-modifying OA drugs (Pontes-Quero+ 2019). Orthobiologic IA injections like mesenchymal stromal cells, platelet-rich plasma, and interleukin-1 receptor antagonist (IL-1Ra) have been recently investigated as biologic therapies to reduce pro-inflammatory markers and inhibit downstream catabolic activity (Van Drumpt+ 2016, Khella+ 2021). One major hurdle with treating OA using IA therapies is the increased rate of clearance from the joint space, with small molecules and proteins having a half-life of ~4 hours (Hunter, D. J. 2015). Thus, there is a need for an intra-articular injection that achieves controlled therapeutic delivery and resides within the joint space for a prolonged period of time. The work herein describes post-synthesis modifications of poly(acrylic acid) (pAA) microgels using lysine and calcium chloride to improve in vitro delivery of IL-1Ra. We hypothesize that the post-synthesis modification of microgels with lysine or calcium will improve sustained release of IL-1Ra through changing electrostatic interactions and/or microgel pore size.

Materials and

Methods: Poly(acrylic acid) micron-sized hydrogels were synthesized and characterized as previously described (Trujillo+ 2024). Microgels were modified with lysine during the wash steps to remove any activated carboxylic acid groups. For the IL-1Ra uptake experiments, dry microgel formulations (4 mg, n=3 per formulation) were first suspended in 1 mL of 0.1 M HEPES and neutralized to pH 7.4 using 1 M NaOH in an Eppendorf tube. Subsequently, 1 mL of 10 mg/mL IL-1Ra was added to each tube and allowed to incubate for 24 hours at 37 ⁰C under stirring at 8 rpm. After 24 hours, microgels were centrifuged and the supernatant was collected. Microgels were washed once by resuspending and centrifuging with 2 mL of PBS to remove any unencapsulated protein. Microgel suspensions with encapsulated IL-1Ra were then either left untreated or treated with 2 mL of 15 mM, 30 mM, or 60 mM CaCl2 for 5 minutes at 4 ⁰C to minimize any additional release of IL-1Ra. Microgels were centrifuged, the supernatant was removed, and microgels were washed once more in 2 mL of PBS to remove any free protein and CaCl2. The release (t=0) started at the initiation of the first wash when the microgels were resuspended in 2 mL of PBS. Half (1 mL) of release supernatant was collected at predetermined time points and replaced with 1 mL of fresh PBS. All supernatants were stored at -20 ⁰C until analyzed. Uptake and release supernatants were analyzed using a BCA Protein Assay Kit or MicroBCA Protein Assay Kit.

Results, Conclusions, and Discussions: IL-1Ra uptake and release were evaluated from the following poly(acrylic acid) microgels formulations: pAA untreated microgels, lysine-functionalized pAA microgels, and pAA microgels treated with 15 mM, 30 mM, or 60 mM CaCl2. IL-1Ra loading capacity was between 94-128% for untreated microgels and the calcium-treated groups, and 51% for the lysine-functionalized microgels (Figure 1). IL-1Ra is mostly negative at physiologic pH, due to its pKa of 5.5. Lysine has a positively charged primary amine at physiologic pH which did not improve the loading capacity of lysine-functionalized microgels compared to other microgel formulations, which have negatively charged carboxylic acid groups (Figure 1). Untreated microgels exhibited burst release of IL-1Ra and released 100% of protein within 6 hours. Lysine-functionalized and calcium-treated microgels reduced the initial burst release and released only 70-88% of IL-1Ra payload over 6 hours. Specifically for calcium-treated microgels, burst release was decreased as the calcium concentration increased, suggesting a dose-dependent response in mitigating IL-1Ra release from anionic microgels. When considering a therapeutic dose of IL-1Ra, in vitro studies show that 100-fold IL-1Ra compared to IL-1 is effective at inhibiting IL-1 activity (Mehta+ 2019, Patwari+ 2009); IL-1ß concentrations in humans with OA have shown to be as high as 0.146 ng/mL (McNulty+ 2013). At each release time point, therapeutic concentrations (100-fold higher than 0.146 ng/mL) of IL-1Ra were delivered for all modified microgel formulations up to 64 days (Figure 2). Positively charged lysine-modified microgels are potentially interacting with negatively charged IL-1Ra and slowing the diffusion of IL-1Ra from the microgel matrix, leading to sustained delivery. We hypothesize that microgels treated with calcium achieve sustained release in two ways: 1.) Calcium decreases the size of the microgel pores after the protein is encapsulated, slowing the burst release and initial rate of diffusion. 2.) Calcium creates ionic interactions with carboxylic acid groups, thus minimizing the ionic interactions of the negative IL-1Ra and negative carboxyl groups and slows the diffusion due to limited charge repulsion. This work demonstrates that simple post-synthesis modifications of polyanionic microgels using amino acids and calcium improves protein release and achieves therapeutic release for greater than 60 days.

Acknowledgements (Optional): These studies were funded by the NSF-GRFP DGE – 2139899 (R.J.T.) and NSF LEAP HI grant (NSF CMMI 2245367). The authors acknowledge the use of facilities and instrumentation supported by the NSF through the Cornell University Materials Research Science and Engineering Center DMR-1719875. We would also like to thank Dr. Alec Macaulay of Cayuga Health for providing the IL-1Ra for our studies.