Treating Chronic Brucella Infections Intracelluar Delivery of Antibiotics Using Novel Hydrophobic Nanospheres

NON-TECHNICAL SUMMARY: During the chronic stage of Brucellosis, the bacteria persist and replicate in tissue resident macrophages. This intracellular niche has proven extremely difficult to target effectively with antimicrobial therapy. Encapsulated antibiotics to the intracellular environment improve intracellular targeting and release of encapsulated antibiotics within Brucella infected cells leading to enhanced bactericidal activity. Targeted intracellular delivery would dramatically increase anti-Brucella activity during the chronic phase of the disease when most patients present with symptoms. It is our hypothesis that antibiotics can be effectively targeted to the specific intracellular niche of various intracellular pathogens using polyanhydride nanospheres. Polyanhydride copolymer nanospheres (PA Nanos) elicit cellular responses from monocytes and dendritic that stimulate internalization, direct intracellular trafficking and degrade slowly within the cells. Varying the chemistry of the particle effects particle degradation and alters the fate of the particle within cells. PA Nanos are capable of entering host cells and delivering antibiotics in the same microenvironment of the pathogen. The highly effective targeting of the intracellular environment greatly reduces the amount of antibiotic needed to treat such an infection and also provided delayed release. We will perform in vivo and in vitro antibiotic efficacy experiments to examine the effect of PA encapsulation on doxycycline antimicrobial activity.

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APPROACH: Intracellular trafficking and fate of nanoparticles is predicted to overlap considerably with Brucella containing vesicles in infected macrophages. This will be conformed using immunofluorescence microscopy to examine the effects of particle chemistry on PA nano internalization, intracellular trafficking and intracellular compartmentalization. These events would have the most significant impact on particle stability, and doxycycline delivery. To understand how variations in nanoparticle chemistry impacts in vivo anti-Brucella activity (specific aim 3), we will perform a series of microscopic experiments to visualize how PA nanos of different copolymer formulations interact with Brucella containing replicative vesicles within murine and human macrophage cell lines. <P>
In order to better understand the impact of doxycycline encapsulation in PA nanos on antibiotic release and on Brucella infections. These included in vitro bacterial survival assays where the number of viable intracellular brucella recovered from monocytes is quantified by colony plating. We will employ the murine monocyte cell lines J774A and RAW264.7 and human THP-1 cells as hosts for B. melitensis infection. We will examine intracellular bacterial survival following 24 hr treatment with PADoxy loaded nanos and quantitatively compare results with infected cultures treated and not treated with equivalent amounts of soluble doxycycline starting at 10 ug/ml. Percent survival of bacteria at 24 and 48 hours were calculated based on the number of internalized bacteria detected at 1 hour post infection which represents 100% of internalized bacteria. Statistical comparisons were made using Student's t-test. Host cell cytotoxicity assays for cultured cells will be recorded using the MTT assay for quantitative measurements and by propidium iodide exclusion for microscopy purposes. <P>
Using information regarding the effective concentrations from SA 1 and 2, mice will be experimentally infected with virulent Brucella abortus strain 2308 (example of chronic infection shown in Figure 3) and treated with PA Nanos by single dose (~10 ug/kg) i.v. tail vein injection at 4 weeks post infection. The status of the chronic infection within BALB/c mice will be measured by harvesting the spleens and livers of mice at 1 week intervals after infection, the tissues will be homogenized in PBS, serially diluted and plated on Schaedler agar plates supplemented with 5% bovine blood. BABL/c mice were chosen as they are regarded as being more susceptible to Brucella infection where the bacterial load in the spleen and liver persists at a much higher level than seen in Blk6 mice. The typical chronic infection, including the "plateau phase" reached at week 2 and extending out to 12 weeks and beyond. Mice are infected i.p. with 1 x 105 CFU in PBS suspension.