Poster X19 - Polymeric Microparticles Loaded with Nanoparticles Promote Sustained and Targeted Delivery of Zoledronate to Activated Macrophages for Osteoarthritis Treatment

Introduction: Osteoarthritis (OA) is a debilitating joint disease marked by cartilage degradation. Despite being a major contributor to functional disability and projected to impact ~1 billion people globally by 2050, there is no cure for OA. Mounting evidence indicates that cartilage debris resulting from joint overuse, injury, or aging often triggers an immune response, leading to macrophage recruitment, activation, and subsequent secretion of proinflammatory and catabolic mediators in the joint. This local inflammation further drives the degradation of remaining cartilage, consequently creating a deteriorating cascade leading to disease progression. Thus, specifically targeting macrophages may hold promise for modulating inflammation, and in effect, slowing OA progression.
Zoledronate (Zol), a potent bisphosphonate drug known for its strong affinity and toxicity towards macrophages, holds potential for abating macrophage activity. However, limited by its hydrophilic and anionic nature, Zol often faces early clearance upon delivery, ultimately hindering its targeting efficiency. To address this limitation, we have developed a novel drug delivery system: nanoparticle in microparticle (NiM) to sustainably deliver Zol to target activated macrophages for OA therapy.

Materials and

Methods: Calcium zoledronate nanoparticles (CaZol-NP) were formed via reverse emulsification technique. CaZol-NP were loaded into polyethylene glycol-polylactic-co-glycolic acid (PEG-PLGA; Sigma Aldrich) microparticles via a coaxial flow phase separation technique to obtain the CaZol-NiM. Encapsulation efficiency of Zol in CaZol-NP and NiM was determined by measuring Zol concentration spectrophotometrically after digesting a known amount of the particles in 5mM PBS (0.1M HCL). The morphology, size, and distribution of CaZol-NP and NiM were determined via transmission electron microscopy (TEM) and optical microscopy respectively, by analyzing sample images with Image J software. Encapsulation of CaZol-NP in microparticles was confirmed using confocal microscopy, Fourier-transform infrared spectroscopy (FTIR), and energy dispersive x-ray analysis (EDAX).
The release kinetics of Zol from CaZol-NP and NiM was determined by incubating the particles in 5mM PBS at neutral and acidic pH (7.4 and 5.0) and the amount of Zol released at different intervals was determined spectrophotometrically. Flow cytometry and confocal microscopy was used to examine cellular uptake of CaZol-NP and NIM by RAW 264.7 (ATCC) macrophages activated with lipopolysaccharides (LPS, Invitrogen). Intracellular localization of the particles was determined by staining cells with lysotracker red DND-99 (Thermo Fisher).
Annexin V apoptosis assay (Thermo Fisher) and MTT test were used to study cytotoxicity of CaZol-NP and NiM using free Zol as control. A mouse model of OA was created by inducing anterior cruciate rupture via cyclic loading of the knee joint and macrophage infiltration was determined by immunostaining.

Results, Conclusions, and Discussions: CaZol-NP and CaZol-NiM were found to have an average size of 63.5 ± 17 nm and 6.9±2 μm respectively (Fig. 1 A, B). The Zol encapsulation efficiency was determined to be ~45% and ~56% for CaZol-NP and CaZol-NiM, respectively. The encapsulation of CaZol-NP in microparticles was confirmed by visualizing the presence of DiD-labelled CaZol-NP in coumarin-6 stained microparticles, (Fig. 1 C). Moreover, EDAX and FTIR analysis of CaZol-NiM showed characteristic peaks for phosphate, confirming Zol encapsulation. Both CaZol-NP and NiM showed higher release of Zol in acidic pH compared to neutral indicating their pH sensitive nature.
Notably, compared to CaZol-NP, CaZol-NiM showed minimal burst release and only 60% of the drug was released in the first 5 days even at the acidic pH (Fig.1D). Cellular uptake studies demonstrated a lower number of CaZol-NP uptake when delivered via NiM compared to free CaZol-NP confirming the sustained release of NP from NiM (Fig.1D).
Further conjugating folic acid to NiM (CaZol NiM-FA) allowed targeted delivery to activated macrophages and both the released CaZol-NP and NiM colocalized with lysosomal compartment upon cellular uptake (Fig 1E). Cell viability in NiM treated samples was significantly higher than the cells treated with free Zol or CaZol-NP (Fig 1F). Also, we identified folate-receptor2 (FR-2)-positive macrophages populations in the OA mouse model.

Altogether, CaZol-NP was synthesized and successfully encapsulated in PEG-PLGA microparticles to obtain CaZol-NiM. The sustained and pH-controlled release of Zol from NiM suggest that CaZol-NiM can allow for spatially targeted delivery only under acidic conditions while minimizing release at physiological pH, thus reducing the risk of side effects of Zol on surrounding tissues. Additionally, identification of FR-2 expressing activated macrophages in mice model of OA, along with successful demonstration of targeted delivery in vitro, can enable specific targeting of CaZol-NiM-FA system to FR-2 expressing macrophages in vivo. Given the critical role of macrophages in OA, CaZol-NiM-FA can provide a potential therapeutic platform for sustained and targeted delivery of clinically translatable bisphosphonates to the OA affected joint while limiting biodistribution to other tissues. Further studies are underway to evaluate in vivo therapeutic efficacy in mice models of OA.

Acknowledgements (Optional): This work is supported by funding provided by the DoD Discovery Award (W81XWH2210020), Institute for Veterans and Military Families (IVMF)- Rostker Dissertation Fellowship Award, and the CUSE- II-17-2020 Award from Syracuse University.