Poster W20 - Engineered Decoy Pretreatment Prevents Phagocytic Clearance of Therapeutic Nanoparticles

Introduction: Polymeric nanoparticles (NPs) are a promising non-viral gene delivery platform for the treatment of genetic diseases. Poly (lactic co-glycolic acid) or PLGA, for example, is a non-immunogenic, biocompatible, and biodegradable polymer that has been used extensively for drug delivery applications and is FDA-approved for several applications [1-4]. One shortcoming of PLGA NPs, however, is that they generally exhibit high accumulation in the liver and spleen after intravenous in-vivo administration, limiting accumulation in extra-hepatic organs [2-4, 5]. This phenomenon is largely due to the activity of the mononuclear phagocytic system (MPS) [6-7]. The MPS refers to a family of phagocytic cells such as macrophages and dendritic cells that are responsible for maintaining homeostasis and clearing foreign objects from blood and tissue [6]. One potential strategy to overcome this clearing is the use of decoys to temporarily occupy or deplete phagocytic cells in the liver and spleen prior to administering therapeutic NPs to increase delivery efficiency to other organs (Figure 1A). We explore two novel decoy pretreatments: Esomeprazole (ESO), an FDA-approved proton pump inhibitor, and unloaded PLGA NPs. Pharmacological and metabolic data have shown that ESO inhibits the liver enzyme cytochrome p450, which is one of the major drug metabolizers in the liver [7-9]. ESO has also been shown to reduce inflammation and consequent macrophage recruitment in a rat sepsis model [10]. Here we show that decoy pretreatment with ESO and unloaded PLGA NPs enables therapeutic polymeric NPs to evade phagocytic clearance

Materials and

Methods: NP Formulation: PLGA NPs were formulated via single emulsion solvent evaporation and loaded with 5 wt% DiD dye. A similar protocol was followed for the formulation of unloaded PLGA NPs; however, different sonication amplitudes were used to achieve a range of decoy NP sizes. A Malvern Zetasizer was used to confirm hydrodynamic diameter and zeta potential of all NPs by dynamic light scattering. In-Vitro Decoy Experiments: RAW 264.7 and NR8383 macrophage cell lines, as well as primary murine bone marrow-derived macrophages were used to study the effects of decoy occupation. For ESO experiments, ESO diluted in DMSO was used to treat cells at a final concentration of 10, 50, or 100 uM for 24 hours. For unloaded decoy PLGA NP experiments, 200, 300, 400, or 500 nm unloaded PLGA NPs were added to cells at 0.05 mg/ml for 24 hours. After the incubation period, cells were treated with 0.05 mg/mL DiD-loaded therapeutic PLGA NPs for an additional 24 hours. Finally, cells were harvested and analyzed by flow cytometry and fluorescence microscopy to quantify cellular uptake of NPs. Media Fluorometry: As a measure of the amount of therapeutic NPs in the media that were not phagocytosed, a sample of the cell culture media was collected at various timepoints throughout incubation with DiD-loaded PLGA NPs and dissolved in DMSO to release the encapsulated dye. A Denovix fluorometer was used to obtain fluorescence measurements of all samples.

Results, Conclusions, and Discussions: Results and

Discussion: Figure 1B depicts the results of an ESO flow cytometry experiment conducted on the RAW 264.7 macrophage-like cell line. Pre-treatment with ESO results in a dual peak phenomenon that is indicative of two different cell populations: one representative of cells substantially clearing therapeutic DiD-loaded NPs and the other representing a population that is only partially clearing DiD-loaded PLGA NPs. With increasing dose of ESO, there is a decrease in percent of cells that are substantially clearing DiD-loaded NPs and an increase in partially NP clearing cells. Figure 1C depicts the results of a blank PLGA NP flow cytometry experiment conducted on the NR8383 alveolar macrophage cell line. We observe a decrease in the percentage of therapeutic DiD NP positive cells with pretreatment of 300, 400, and 500 nm blank PLGA NP decoys. Similar experiments with primary murine bone marrow-derived macrophages are currently ongoing.

Conclusions: Together, these data suggest that both ESO and unloaded PLGA NP decoys can decrease phagocytic clearance of therapeutic PLGA NPs, i.e. fewer therapeutic NPs are sequestered by occupied macrophages. This strategy is designed to increase NP trafficking to tissues other than the liver and spleen, such as the lungs, enhancing in vivo delivery efficiency of therapeutic polymeric NPs. Dosing and selection of decoys will need to be optimized depending on the application and assessed for safety. In conclusion, we find that treatment with decoys prior to therapeutic NP administration evades phagocytic clearance, theoretically allowing for increased in vivo delivery to multiple organs.

Acknowledgements (Optional): This work was supported by grants from the National Institutes of Health (NIH; R00 HL151806), the Cystic Fibrosis Foundation (CFF; PIOTRO21F5, HASTIN23G0-COLLAB), and Emily’s Entourage (to A.S.P.).

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