(J-380) Alginate-Lysozyme Nanogels as Neutrophil-Targeting Aminoglycoside Antibiotic Delivery Vehicles

Introduction:: Neutrophils are first responder cells in acute inflammation and infection and are the primary killers of microbes in bacterial infections. Recent work in our group has shown that protein nanogels have tropism for neutrophils that accumulate in the lungs in acute inflammatory models of sepsis. Saccharide-protein nanogels had especially good tropism for inflamed lungs. Here, we devise an alginate-lysozyme saccharide-protein nanogel, with the goal of complexing the negatively charged alginate with aminoglycoside antibiotics. The nanogels comprise an agglutinated lysozyme core and a hydrophilic alginate shell (Figure 1a) that can retain drug cargo with positive or partial positive charge like aminoglycosides (Figure 1b-c). Since similar nanogels target neutrophils in sepsis models, these new nanoparticles could be used to deliver antibiotics specifically to sites of acute bacterial infection by accumulating in the cells most involved in fighting those infections. These studies are designed to demonstrate synthesis of alginate-lysozyme nanogels, formation of stable complexes of the nanogels with amikacin, delivery of the nanogels to neutrophils in vitro, and biodistribution data for targeting the nanogels to inflamed/septic lungs.

Materials and Methods:: Alginate-lysozyme nanogels (ALNGs) were synthesized via Maillard conjugation of alginate to lysozyme (1:2 mol:mol) followed by heat gelation of the alginate-lysozyme conjugates at the lysozyme isoelectric point. ALNGs were characterized with nanoparticle tracking analysis, dynamic light scattering, and zeta potential analysis. ALNGs were incubated in solutions of amikacin at different concentrations to load with antibiotic. Excess amikacin was removed from amikacin-ALNGs by centrifugation. Excess amikacin was quantified by derivatization with pentanedione and formaldehyde, forming optically active lutidine groups, allowing subtractive quantification of amikacin loading efficiency. Amikacin-ALNGs were characterized for size, concentration, and zeta potential, as with ALNGs. Mouse neutrophils were isolated from femoral bone marrow using the EasySep magnetic neutrophil isolation kit. ALNGs or amikacin-ALNGs were labeled with succinimidyl ester Alexa Fluor 647, pre-incubated with mouse serum, then incubated with neutrophils. After rinsing away unbound ALNGs, neutrophils were stained with PerCP-Cy5 anti-Ly6G. ALNG uptake in neutrophils was assessed with flow cytometry. ALNGs were labeled with Iodine-125 for tracing in biodistributions. Distribution of ALNGs to different organs was assessed 30 minutes after intravenous injection in naïve mice, mice receiving intravenous bacterial lipopolysaccharides, or mice with sepsis induced by cecal ligation and puncture. Distribution of ALNGs to different organs after inhaled delivery in naïve mice was assessed at 1 hour, 3 hours, 6 hours, and 24 hours after inhalation.

Results, Conclusions, and Discussions:: Alginate-lysozyme nanogels (ALNGs) have a mean diameter of ~90 nm and an average Zeta potential <-35 mV. ALNGs are stable for months in suspension at 4°C. Amikacin was osmotically loaded in ALNGs at an efficiency of ~50% up to amikacin:ALNG mass:mass 1:1. Amikacin was loaded in ALNGs at >50% of ALNG mass (Figure 2a). Testing for amikacin leak from ALNGs over two weeks storage at 4°C, we found <5% loss of loaded drug. ALNG size distributions were not significantly changed by addition of amikacin, indicating that charge-based amikacin-ALNG complexing did not cause substantial aggregation (Figure 2b). Zeta potential measurements confirmed electrostatic interactions between amikacin and ALNGs. Amikacin shielded the negatively charged alginate, leading to increase in the zeta potential as amikacin loading concentration increased (Figure 2c). Flow cytometry testing of ALNG and amikacin-ALNG uptake in neutrophils showed; a) ALNGs are taken up in neutrophils; b) Amikacin loading increases ALNG uptake in neutrophils, as measured by fraction of neutrophils taking up ALNGs (~50% increase) and mean ALNG fluorescence in neutrophils (~200% increase) (Figure 3). Acute injuries modeling sepsis lead to increased uptake of ALNGs in the lungs (Figure 4a). An ~40% increase in lung uptake was measured for model sepsis induced by both intravenous lipopolysaccharides and cecal ligation and puncture. Previous work shows that saccharide-protein nanogels have tropism for inflamed lungs due to uptake in neutrophils. Finally, inhaled ALNGs focally retained in the lungs and oropharynx (Figure 4b), with retention in the oropharynx maintaining a steady concentration over 24 hours. ALNGs are therefore a new nanoparticle designed to stably retain large quantities of positively charged drugs, as demonstrated for the aminoglycoside antibiotic amikacin. ALNGs have tropism for neutrophils that is enhanced by loading with amikacin. ALNGs are selective for the lungs in sepsis, likely driven by uptake in neutrophils. Our data indicate that ALNGs may achieve selective delivery of antibiotics to the lungs, site of many acute infections, and to neutrophils the innate immune system fighters of infections.

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