U42 - Highly Selective Targeting of Circulating Neutrophils for Cellular Drug and Gene Delivery

Introduction: : Synthetic nanoparticles (NPs) have been extensively explored for targeted drug delivery principally due to their preferential accumulation in the site of diseases. Despite acquiring some FDA approvals, further clinical application of nanoparticles is majorly hindered by their limited ability to overcome biological barriers resulting in uncontrolled biodistribution and high clearance. Cell hitchhiking has been emerging to overcome these challenges since they are inherently biocompatible and have improved access to tissues and organs. Immune cells are ideal for hitchhiking because they target various diseases naturally. Neutrophils are the most abundant circulating immune cells in humans, playing a central role in acute inflammation. Adhesion to vasculature and tissue infiltration of neutrophils are key processes in acute inflammation. Many inflammatory/autoimmune disorders and cancer therapies have been found to be involved in activation and tissue infiltration of neutrophils. Neutrophils must be activated before they can ingest foreign nanoscale therapeutics that are present in circulation. Very few approaches have been reported to selectively activate neutrophils in situ. External stimuli were once used to generate local inflammation only at the disease site to activate passing neutrophils that can engulf a limited number of nanoparticles before infiltrating to the problematic tissue. Pathogen-mimicking nanoparticles were elaborately designed to take advantage of the intrinsic ability of neutrophils to detect and take up pathogens via the recognition of pathogen-associated molecular patterns. Here we report a convenient method that uses fusogenic liposomes to selectively activate circulating neutrophils systematically which then engulf nanoparticles.

Materials and

Methods: : Two types of liposomes, one fusogenic version for the selective activation of neutrophils and the other, the classic stealth version as a model of nanoscale therapeutics, were prepared using the hydration/extrusion method. To conjugate E-selectin to liposomes, a stoichiometric amount of his-tagged protein was added to pre-formed liposomes. Human blood was collected from fully consented healthy donors. To test the activation and uptake of nanoparticles of neutrophils, fusogenic liposomes and stealth liposomes were added to 500 µL blood at a liposome/leukocyte ratio of 1,000 to 100,000 assuming the leukocyte count is 7,000/µL. After incubation, neutrophils and mononuclear leukocytes were isolated using PolymorphPrep (Progen). The cells were analyzed with confocal microscopy (LSM 900, Zeiss) and flow cytometry (Guava EasyCyte HT, Millipore Cytek). A transmigration assay was used to test the migrating ability of neutrophils before and after activation. Briefly, transwell inserts were coated with Type I collagen. 1 million neutrophils were plated in each transwell well on top of the collagen layer. fMLP was added to the bottom of the transwell system as a chemotaxis attractant to initiate migration. Cells in the bottom of transwell were enumerated from bright field images. BALB/cJ mice were used to test the activation of neutrophils in vivo. Fusogenic liposomes and stealth liposomes were administered from the tail vein at the same liposome-to-leukocyte ratio as above. Blood was collected via cardiac puncture at predetermined time intervals to isolate neutrophils for the analysis of activation, content of stealth liposomes and migrating ability.

Results, Conclusions, and Discussions:: The classic fusogenic liposome formulation contains nearly 50% positively charged lipid DOTAP, which is responsible for their toxicity and has held them from being used in vivo. We incorporated phase separation in the formulation to concentrate DOTAP in the small phases of liposomes and reduced its’ content by >10 folds (Figure A). E-selectin (ES) is an adhesion protein that preferably adheres to neutrophils over other major subpopulations of leukocytes. We conjugated ES to the surface the fusogenic liposomes to increase their selectivity to neutrophils over other subpopulations of leukocytes. The increase in size distribution of the liposomes after E-selectin conjugation supports the association of the protein to the surface of liposomes. A 15 min incubation of ES-conjugated fusogenic liposomes in whole blood was enough to activate all the neutrophils. In sharp contrast, liposomes were not found in or associated with mononuclear leukocytes including monocytes and all the subpopulations of lymphocytes, as well as red blood cells (Figure 1B). The activation and internalization of liposomes were also found dependent on the number of ES conjugated on the surface of fusogenic liposomes, with more ES showing higher uptake of the liposomes (Figure 1C). In the transendothelial migration assay, no impairment in the migrating ability of neutrophils activated by the fusogenic liposomes was found as compared to non-treated resting neutrophils. Animal studies were carried out to test the potential of ES-conjugated fusogenic liposomes to selectively activate neutrophils and engulf stealth liposomes as a model of common nanoparticles. We report a convenient method for the activation of neutrophils in situ. The activation is surprisingly specific, which lays down the foundation for loading nanoparticles-encapsulated therapeutics into neutrophils for tissue specific drug delivery. Stealth liposomes are arguably the most successful drug carriers that can deliver a wide range of therapeutics including small molecules, DNA/RNA and proteins. Hitchhiking neutrophils with drug loaded stealth liposomes thus hold great potential for the treatment of various diseases.

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