We hypothesize that the targeted delivery of the immune agonist poly(I:C) to macrophages and dendritic cells in the ovarian tumor environment can synergize with chemotherapy and immune checkpoint blockade to lead to the production of inflammatory cytokines, increased antigen presentation and effector T cell infiltration in the tumor to promote long-lasting anti-tumor immunity. To achieve this, we determined optimal nanoparticle physiochemical properties that promote association with immune cells, developed a LbL NP encapsulating poly(I:C) for targeted delivery, and evaluated the effectiveness of our targeted NP therapy in a syngeneic model of ovarian cancer.
Results: Due to the modular nature of LbL, we optimized the 1) cell-targeting surface chemistry by varying the negatively-charged terminal layer, 2) the stiffness of the liposomal core to promote NP internalization, and 3) drug loading of poly(I:C).
To design drug carriers that direct therapeutics to specific cells, we elucidated how the surface chemistry and core composition of Layer-by-Layer nanoparticles (LbL NPs) influences association of particles with ovarian cancer and antigen-presenting cells (APCs). LbL NPs formulated with varying surface chemistries—including glycan-decorated polymers and native polysaccharide polymers—were screened on ovarian cancer, RAW264.7 macrophage-like cells and primary dendritic cells (DCs) to identify candidates with high NP-cell association. This revealed that dextran sulfate enhanced NP association with macrophages. Contrarily, our novel galactose-decorated polymer enhanced targeting to DCs, likely due to interactions with galactose-binding lectins expressed on DCs. LbL NPs with softer liposomal cores showed enhanced intracellular accumulation in APCs compared to rigid cores.
We then developed a LbL NP loaded with Poly(I:C) which is a potent TLR-3 immune agonist that has poor half-life and toxicity profiles as a free drug, necessitating the use of a drug carrier. The Poly(I:C)-loaded NPs were formulated with the optimal physiochemical properties—soft liposomal core and dextran sulfate outer layer—that were identified to promote delivery to APCs. Poly(I:C) was loaded into the LbL NPs via direct adsorption as one of the polyelectrolyte layers as Poly(I:C) has an inherent negative charge, enabling it to function as a layer. The assembly conditions were optimized to yield stable NPs with a loading of nearly a 1:1 weight ratio of poly(I:C) to liposomal core. The targeted Poly(I:C) LbL NPs facilitated greater poly(I:C) delivery to macrophages and DCs, resulting in nearly a 4-fold upregulation of CD86 expression and 2-fold TNF-a cytokine production compared to free poly(I:C) in vitro
In a syngeneic model of ovarian cancer, we demonstrate that the optimized Poly(I:C) LbL NP increases the half-life of poly(I:C) when administered intraperitoneally compared to poly(I:C) delivered without a carrier and enhances TNFa cytokine production in vivo. Additionally, the Poly(I:C) LbL NP synergizes with chemotherapy drugs capable of inducing immunogenic cell death (ICD)—oxaliplatin and doxorubicin—to reduce tumor growth and promote longer survival.
This work demonstrates the targeted delivery of immunotherapeutic agents using LbL NPs with optimized physiochemical properties can dramatically improve the delivery of an immune agonist to APCs for a more specific and effective immunotherapeutic treatment strategy.