In-Space Production of Janus Base Nanoparticles Enhances mRNA Delivery for the Treatment of Osteoarthritis

Introduction: :

Introduction: Osteoarthritis (OA) is a degenerative joint disease affecting over 32.5 million adults in the U.S., characterized by progressive cartilage breakdown, pain, and impaired mobility. Despite the prevalence and burden of OA, there are currently no FDA-approved disease-modifying therapies. Protein-based drugs such as Anakinra, an IL-1 receptor antagonist, have shown limited efficacy in OA due to poor joint retention and rapid degradation. mRNA-based analogs offer an alternative strategy by enabling intracellular production of therapeutic proteins, but their clinical translation is constrained by significant delivery barriers. Effective mRNA delivery to chondrocytes is limited by two primary obstacles: (1) the dense extracellular matrix (ECM) of cartilage, with pore sizes averaging ~20 nm, and (2) endosomal entrapment following cellular uptake. Conventional lipid nanoparticles (LNPs), typically spherical and >50 nm in diameter, exhibit poor ECM penetration, low cartilage retention, and cytotoxicity. To address these limitations, we developed Janus Base Nanoparticles (JBNps)—a new class of self-assembled, rod-shaped nanostructures inspired by DNA base pairing. JBNps exhibit a slim anisotropic morphology that enables cartilage penetration and early endosomal escape via transient pore formation, promoting efficient cytosolic mRNA release. Their non-covalent, DNA-mimetic chemistry ensures low toxicity, high structural stability, and biocompatibility. Building on this design, we further explore the benefits of in-space manufacturing. Microgravity conditions enhance JBNp self-assembly, uniformity, and functional performance. This work presents the potential of space-fabricated JBNps to improve mRNA delivery for OA treatment and highlights the emerging role of microgravity in advancing precision nanomedicine.

Materials and

Methods: : Materials and

Methods: Enhanced cartilage penetration of rod-shaped JBNp was demonstrated by transfecting bovine cartilage explants with Cy5 mRNA using JBNp. Therapeutic efficacy was tested in a murine destabilization of the medial meniscus (DMM) model of osteoarthritis. JBNp-IL1Ra-encoding mRNA were administered via intra-articular injection, and joint function was evaluated using behavioral assays including thermal and mechanical sensitivity tests, gait analysis, and paw area measurements. Cartilage integrity and osteophyte formation were assessed via micro-computed tomography (micro-CT) and histological staining. The extracted knee joints following this treatment regimen were imaged via Micro-CT, and sections were stained for Safranin O/Fast Green. Behavioral studies were also conducted, including thermal sensitivity (hot plate), mechanical sensitivity (Von Frey) analysis, and gait analysis.

To evaluate the impact of microgravity on JBNp fabrication, in-space assembly and mRNA loading were performed aboard the International Space Station (ISS) during the SpaceX CRS-30 and CRS-31 missions. Samples were returned to Earth for analysis of morphology via TEM, size distribution, and drug loading efficiency via UV-Vis spectroscopy and fluorescence imaging. In vitro transfection efficiency and mRNA bioactivity were assessed in mammalian cell cultures, while in vivo functionality was evaluated in murine models following ambient storage.

Results, Conclusions, and Discussions::

Results: In the bovine cartilage explant model, JBNps penetrated deeper and more efficiently than conventional LNPs (Fig 1B), enabling efficient mRNA delivery through the dense ECM. In the DMM-induced murine OA model, intra-articular administration of JBNp-IL1Ra mRNA resulted in significant improvements in thermal and mechanical sensitivity, increased stride length, and enlarged paw contact area (Fig 1C). Micro-CT imaging revealed reduced osteophyte formation, while histological analysis showed preservation of proteoglycan-rich cartilage and decreased expression of degradation markers (Fig 1D).

JBNps assembled under microgravity aboard the ISS displayed enhanced structural uniformity, thinner dimensions, and greater mRNA loading capacity compared to Earth-fabricated controls (Fig 2A-D). These in-space–produced nanoparticles maintained high bioactivity after 45 days of ambient storage and exhibited improved transfection efficiency in vitro (Fig 2E). In vivo studies using aged, space-assembled JBNps further demonstrated superior therapeutic outcomes in OA mice, suggesting that microgravity conditions can enhance JBNp performance and stability (Fig 2F). Together, these findings support the use of JBNps as a dual-platform therapeutic system with terrestrial efficacy and space-enabled manufacturing advantages.

Discussion and

Conclusions: JBNps represent a transformative advancement in mRNA delivery for osteoarthritis therapy, addressing critical limitations of existing nanoparticle platforms. Engineered with DNA-inspired, rod-shaped nanotube bundles, JBNps efficiently penetrate dense cartilage extracellular matrix and demonstrate superior intracellular mRNA delivery through early endosomal escape via transient pore formation. This capability translates into sustained therapeutic mRNA bioactivity, enhanced cartilage retention, and significantly reduced cytotoxicity and immunogenicity. Importantly, JBNps are uniquely suited for space-based manufacturing, as evidenced by experiments aboard the International Space Station. Microgravity significantly enhances JBNp assembly, resulting in improved structural uniformity, increased mRNA loading efficiency, and enhanced therapeutic efficacy both in vitro and in vivo. In an OA mouse model, intra-articular injection of JBNp-IL1Ra mRNA effectively reduced cartilage degradation and improved thermal and mechanical sensitivity. Beyond OA, JBNps’ adaptability positions them as a versatile platform for delivering a wide variety of therapeutic RNAs and small-molecule drugs, including those targeting solid tumors. Overall, this study highlights the promise of JBNps as an innovative therapeutic vehicle with potential applications spanning terrestrial medicine and in-space biomanufacturing.

Acknowledgements and/or References (Optional)::