Abstract
In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery.
Highlights
Uptake may be extremely selective, as many invasive pathogens such as Salmonella, Yersinia, and reovirus exploit specific mechanisms of M cell transcytosis
Development and Physiochemical Characteristics of poly(lactic-co-glycolic acid) (PLGA) Nanoparticles with plx—We showed previously that claudin 4-targeted protein incorporated into PLGA nanoparticles can mediate M cell-targeted delivery [24]
Mucosal epithelium M cells are an important entry point for many invasive pathogens, so we investigated zeta potential and M cell uptake of bacterial strains S. aureus, S. pneumoniae, and Yersinia enterocolitica (Yersinia), which are prevalent in the mucosal immune system, and shown to be taken up by M cells
Summary
Materials—The PLGA (poly(DL-lactide-co-glycolide) 85:15, molecular weight 50,000 –75,000), polyvinyl alcohol (molecular weight 30,000 –70,000, 87–90% hydrolyzed), and plx (molecular weight: 8500) were obtained from Sigma-Aldrich. 1 M HEPES, phosphate-buffered saline (PBS, 1ϫ), SDS solution (10%) and Staphylococcus aureus (Wood strain without protein A) BioParticles-Alexa Fluor 488 conjugate were purchased from Invitrogen. Samples of PLGA nanoparticle dispersion in PBS (1 mg/ml concentration) were placed in a disposable cuvette for size measurements. Each sample was measured three times for triplicate preparations of nanoparticles and is reported as mean Ϯ S.D. Determination of Total Protein Loading (%w/w) and Surface Loading—Total protein loading was estimated using a BCA assay. Blank PLGA nanoparticles were prepared, and protein loading for these “nonprotein-loaded particles” was measured using BCA assay. 5– 8 mg of freeze-dried nanoparticles from three different experiments were measured accurately and dispersed in 1 ml of 2% SDS solution. The zeta potentials of bacterial strains Staphylococcus aureus, Streptococcus pneumoniae, and Yersinia were measured in different ionic strength PBS solutions by dispersing 8 ϫ 107 bacteria per 1.5 ml of solution. Because of the higher rate of uptake of bacteria, counts were measured using 1600 m2 areas, and means Ϯ S.E. for the number of particles taken up in each was plotted for three independent experiments for each condition
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