(184m) Microfluidic Assembly of Layer-By-Layer Nanodiscs for the Targeted Delivery of IL-12 to Ovarian Cancer

Ovarian cancer is a severe disease projected to result in over 20,000 new diagnoses in the US in 2025 and is one of the most difficult to detect and treat, resulting in it being among the deadliest for women. It is typically diagnosed in late stages in which 5-year survival rates remain below 40% under the current standard-of-care of surgical resection and platinum/taxane-based chemotherapeutics. Moreover, ovarian cancers frequently develop resistance to chemotherapies, leading to aggressive, metastatic recurrences in about 70% of patients. Immunotherapy is an alternative treatment class gaining clinical relevance due to its ability to stimulate the patient’s own immune system to recognize and eliminate cancer cells. One immunotherapeutic of particular interest in the context of ovarian cancer is interleukin-12 (IL-12), a potent cytokine that activates natural killer cells and T cells and induces the production of interferon-gamma (IFN-γ), a downstream cytokine that promotes an antitumor response. However, treatment with free IL-12 has been precluded from the clinic due to the systemic toxicity of a therapeutically relevant dose. Nanoparticles are well-positioned to fill this therapeutic gap by providing a platform upon which to anchor and deliver potent immunostimulatory molecules directly to the tumor microenvironment while minimizing off-target effects.

Nanodiscs are an intriguing lipid-based nanoparticle comprised of a single discoidal lipid bilayer and an edge typically stabilized with steric moieties such as PEG or membrane scaffold proteins. These nanoparticles are characterized by a high aspect ratio (less than 50 nm in diameter, approximately 8 nm in thickness) and a hydrophobic core. Recently, a new class of nanodiscs has emerged from our lab where the disc’s edge is stabilized by a charged lipid headgroup. In mouse models, Layer-by-Layer (LbL) charge-stabilized nanodiscs (CNDs) have demonstrated enhanced tumor accumulation and penetration compared to spherical liposomes due to their smaller physical size and anisotropic shape. In this work, we show that LbL-CNDs can be produced via microfluidic assembly to improve the reproducibility and scalability of this promising therapeutic platform to enhance its translational potential.

Commercially available microfluidic chips were used to synthesize the CND core. A microfluidics-based approach is preferred to the previous detergent dilution-based method due to the reduction of batch-to-batch variability and increased control over the parameters impacting particle size (i.e., total flow rate and flow rate ratio). The surface of the CNDs is then decorated with covalently-anchored IL-12 molecules via maleimide-thiol conjugation chemistry. The anionic (IL-12)-CNDs are subsequently layered with a supporting polycationic polymer followed by a polyanionic polymer that has demonstrated affinity for ovarian cancer. LbL-(IL-12)-CNDs are then tested for IL-12 bioactivity, immunostimulatory capacity, and tumor association in vitro. The combination of covalent linkage to our nanocarrier and tumor-specific nanoparticle coating serves to minimize the risks of systemic IL-12 toxicity while allowing for a targeted and potent dose of cytokine directly to the ovarian tumor microenvironment. This work establishes a scalable microfluidics-based platform to synthesize nanoparticles for the targeted delivery of IL-12, offering a promising strategy to enhance immunotherapy efficacy while mitigating systemic toxicity in ovarian cancer treatment.