Intestinal uptake of Polymyxin B microparticles

Introduction Polymyxin B (PMB) is a cationic peptidic antibiotic used extensively for parenteral and topical treatment of Gram-negative infections. However, PMB therapy is associated with considerable toxicity, mainly nephrotoxicity and neurotoxicity. The oral route for PMB administration has not been considered owing to the pH value and proteolytic degradation in the gastrointestinal tract, as well as the negligible absorption through the intestinal absorption, probably owing to its cationic charge. Therefore, to achieve a PMB oral administration perspective, providing less toxicity and cationic charge, calcium alginate/chitosan microparticles were designed. The developed microparticles, previously characterized in vitro and in vivo, showed clear advantages in terms of reduced PMB toxicity and prolonged systemic antibacterial effects [1-3]. To examine the microparticle uptake process, in the present study, cell internalization capacity of fluorescent microparticles loaded with PMB was evaluated by means of Caco-2 cell lines and quantified by cytofluorimetry. Moreover, microparticle location inside the cells was revealed by confocal microscopy. Experimental methods Microparticle preparation The microparticle preparation and the in vitro characterisation were previously described [1]. Shortly, fluorescent calcium alginate/chitosan (CaA/CHT/FITC) microparticles were prepared by spray-drying a 0.5% water solution of NaA/PMB/FITC (3:1:0.01) and by crosslinking the microparticles with calcium chloride and chitosan. The obtained crosslinked microparticles were characterised for morphology and size, fluorescence, FITC release in simulated intestinal fluid, PMB content, PMB in vitro release and microbiological activity preservation. Caco-2 internalization Caco-2 cells were cultured at 37°C in Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM) with 10% FBS (Foetal Bovine Serum) and 1% nonessential aminoacids. Cells were seeded 48 h before the incubation with the microparticle suspension. Fluorescent FITC crosslinked microparticles suspended in water (10 mg/5 ml) and diluted (1:1 or 1:2) in DMEM, were applied to the cell monolayer and then incubated at either 4°C or 37°C for 5 h or overnight. Cells were washed twice with Phosphate Buffer Saline (PBS) and then collected by trypsinization for the cytometry analysis or alternatively fixed for confocal microscopy. Flowcytometry evaluation of intracellular uptake was performed by a Coulter Epics XL flow cytometer equipped with 488 nm argon laser. FITC fluorescent cells were expressed as a percentage of the total cell population. Results and discussion As previously observed [1, 2], the obtained CaA/CHT/FITC microparticles exhibited a good water dispersibility, a nearly spherical shape with a wrinkled surface (Figure 1) and a size ranging from 0.5 to 2.5 µm (mean size 0.78 ± 0.55), being about 75% of population less than 1 µm. Moreover, microparticles were still fluorescent following a 4 h release assay in simulated intestinal fluid, as observed by epifluorescence videomicroscopy (Figure 2). Figure 1 – SEM image of CaA/CHT/FITC microparticles Figure 2 - Epifluorescence microscopy image of CaA/CHT/FITC microparticles The antibiotic entrapped inside the microparticles (loading level 11.86 ± 0.70% w/w, encapsulation efficiency of about 47%) which was found associated to the alginate chain by an electrostatic interaction and maintaining its microbiological activity, was released negligibly in simulated gastric fluid at pH 3.0 and gradually in simulated intestinal fluid. Uptake and absorption of CaA/CHT microparticles in Caco-2 monolayers were evaluated by cytofluorimetry and confocal microscopy. Fluorescence microscopy revealed that the microparticles were indeed attached to Caco-2 cells following 5 h incubation. Reducing the temperature from 37°C to 4°C resulted in a reduction in the number of microparticles associated with Caco-2, suggesting an internalization process which is temperature dependent, i.e. an intracellular location of the microparticles. This finding was confirmed by confocal microscopy showing fluorescent spots at different depths from the apical surface, attributable to the microparticles. Figure 3 – Cytofluorimetric analysis in function of a) concentration, b) temperature, c) time Conclusions The CaA/CHT microparticles were internalized in CaCo-2 cells, therefore the system could be useful in perspective of an oral PMB administration.