Development of particulate drug formulation against C. parvum: Formulation, characterization and in vivo efficacy.

This research aims towards developing an alternative therapy against Cryptosporidium parvum using bioadhesive paromomycin and diloxanide furoate-loaded microspheres. Microspheres were prepared using chitosan and poly(vinyl alcohol) and two types of cyclodextrins (ß-CD and DM-ß-CD) for the potential use of treating cryptosporidiosis. This pathogen is associated with gastrointestinal illness in humans and animals. Microparticle formulations were characterized in terms of size, surface charge, drug release and morphology. In vivo bioadhesion properties of CHI/PVA microspheres were also evaluated in mice. Finally, the in vivo efficacy of CHI/PVA microspheres against C. parvum was tested in neonatal mouse model. In this work, microspheres prepared by spray-drying showed spherical shape, diameters between 6.67±0.11 and 18.78±0.07µm and positively surface charged. The bioadhesion studies demonstrated that MS remained attached at +16h (post-infection) to the intestinal cells as detected by fluorescence. This finding was crucial taking use of the fact that the parasite multiplication occurs between 16 and 20h post-infection. The efficacy of treatment was determined by calculating the number of oocysts recovered from the intestinal tract of mice after 7days of post-infection. Mice receiving orally administered microspheres with and without drug exhibited significantly lower parasite loads compared with the control mice. Ultrastructural observations by TEM bring to light the uptake of smallest particles by enterocytes associated with conspicuous changes in enterocytic cells. Completely recovery of cell morphology was detected after 24h of first inoculation with MS. CHI/PVA microspheres appear to be a safe and simple system to be used in an anticryptosporidial treatment. The distinctive features of neonatal mice requires further work to determine the suppressive effect of this particulate delivery system on C. parvum attachment in other animal models.

In vitro evaluation of the suppressive effect of chitosan/poly(vinyl alcohol) microspheres on attachment of C. parvum to enterocytic cells.

We present a new strategy to suppress the attachment of Cryptosporidium parvum to the enterocytes cell surface by bioadhesive microspheres. An optimized microsphere system based on chitosan/poly(vinyl alcohol) was prepared by experimental design for the delivery of Diloxanide Furoate-cyclodextrin complex. Formulations were characterized in terms of size, surface charge, drug release, IR spectroscopy and morphology. Bioadhesion properties of chitosan/poly(vinyl alcohol) microspheres, evaluated in the human enterocytic HCT-8 model, were concentration and time dependent. In vitro efficacy of chitosan/poly(vinyl alcohol) microspheres against Cryptosporidium was tested in infected cultures and stages of parasite were assessed by immunofluorescence. The degree of adherence to cells and the inhibition of infectivity were directly related with the lowest level of cross-linking. The C. parvum attachment to cells surface was efficiently suppressed by a concentration of 100 µg/ml of microspheres. TEM observations showed no epithelial-cell damage when microspheres were co-incubated in infected cultures. These results were coincident with the lack of toxicity in cytocompatibility studies. Microspheres remained adhered after 72 h to the apical area of enterocytes. The results suggest that chitosan/poly(vinyl alcohol) with adequate size and appropriate surface characteristics suppress by impairment the attachment of sporozoites to enterocytes and may have a great potential in the oral chemotherapy of Cryptosporidium infections.