(489e) Formulation, Efficacy, and Delivery of Organic Nanocarriers Encapsulating Streptomycin for Treating Trees Infected with Citrus Greening Disease

Background: Citrus greening disease, caused by the phloem-limited Candidatus Liberibacter asiaticus (CLas), is one of the most destructive diseases of citrus worldwide, with currently no effective management strategies or known resistances. Antibiotics, such as streptomycin and oxytetracycline, have been shown to reduce CLas titer in infected citrus trees following trunk injection; however, the uptake and subsequent phloem-loading of these antibiotics in a practical, time-efficient, and widely used manner (i.e., via foliar spraying) has been largely unsuccessful with currently available products, even in the presence of uptake adjuvants. In a previous study, organic-coated nanocarriers (NCs) were shown to be phloem-loaded and translocated systemically following foliar administration in tomato plants, motivating the development of surface-similar NCs encapsulating antibiotics for targeted delivery to the phloem of citrus greening-infected trees.

Methods: Encapsulation of streptomycin (STP) into organic NCs with biocompatible surfaces (lecithin, cellulose, and sucrose) was achieved via Flash NanoPrecipitation (FNP) coupled with hydrophobic ion pairing using different counter ions (sodium oleate, dodecyl sulfate, and dodecylbenzosulfonate) and a hydrophobic co-core of vitamin E acetate (Inset 1 in Figure 1). Encapsulation efficiency (EE) and in vitro release of STP from the NCs in simulated apoplastic fluid (SAF) over 14 days was quantified via HPLC. The efficacy of the NCs against L crescens, a culturable analog of CLas, was assessed via optical density (OD) assays (Inset 2 in Figure 1). Lab-scale uptake and translocation (Inset 3 in Figure 1) of surface-similar NCs containing dye or metal in citrus plants was assessed via confocal microscopy and Inductively Coupled Plasma Mass Spectrometry (ICP-MS), respectively. Scale up of NC production was accomplished using syringe pumps and mass flow controllers.

Results and Implications: Streptomycin was encapsulated at high efficiency (EE > 96%) in a variety of nanocarrier formulations with a range of sizes (70 to 250 nm) and STP concentrations (125 to 1000 ppm). Different counterions, charge ratios, and presence of co-core provided controlled in vitro release of STP from NCs in SAF varying from 2 to 50% of the theoretical maximum over two weeks. Low release of STP from the NCs is an important first step to demonstrate that STP stays associated with the NC, and hence, would reach the same plant tissues as the NCs do after application. Lab-scale translocation studies done on small (50 cm height) citrus trees have shown that lecithin-coated NCs encapsulating a metal probe were the only ones that reached the root tissues following foliar spraying. Approximately 7% of the NCs detected were found in the roots and 33% in the stem, providing an estimated NC concentration in the phloem of these tissues of 2 to 10 ppm (equivalent to 0.4 to 2 ppm of STP). Additionally, all NC formulations were as efficacious against L crescens as free STP (at concentrations ranging from 500 to 0.05 ppm) in vitro over 7 days, demonstrating no loss of antibiotic activity due to encapsulation. These preliminary results are a promising indication of the potential of encapsulated STP to be effectively delivered to the phloem of citrus-greening infected trees to combat CLas. A year-long field trial on 5-year-old OLL and Hamlin citrus cultivars is underway to test the performance of the best STP NC on actively diseased trees (Inset 4 in Figure 1).