We previously reported that some forms of bovine lactoferrin (bLf) are effective in substantially delaying C. difficile growth and preventing production of toxins in a human in vitro gut model of C. difficile infection (CDI). The aim of the present study was to develop lactoferrin-loaded alginate microparticles coated with high molecular weight chitosan for enhanced protein stability, and to subsequently evaluate their anti-C. difficile defence properties in vitro.Methods
Different forms of bLf (iron-depleted; apo-bLf, iron-saturated; holo-bLf, and manganese-saturated; Mn-bLF) and BSA-FITC were encapsulated in calcium-alginate and chitosan-coated alginate particles. Microgel particles were fabricated using the emulsification/internal gelation method. Protein encapsulation efficiency was confirmed by fluorescence microscopy imaging of BSA-FITC-loaded hydrogel particles. In vitro release studies conducted in pH-simulated gastrointestinal conditions were employed to investigate protein-loading efficiency and release rate of encapsulated protein. The various encapsulated bLf forms were evaluated for their influence on intestinal epithelial barrier function and cell viability alone, and in combination with purified whole C. difficile toxins A and B or bacterial supernatant samples of the epidemic 027 C. difficile strain. Enterocyte viability and epithelial permeability were assessed using trypan blue exclusion, MTT cytotoxicity assay and changes in trans-epithelial electrical resistance (TEER) in Caco-2 cells, respectively.Results
Alginate microparticles are suitable for encapsulation and pH-triggered release of metal-bound bLF proteins. The application of bLf (5 mg/mL) delivered from alginate microparticles to human intestinal epithelial cells (hIECs) significantly reduced the cytotoxic effect of toxin A and bacterial supernatant samples on Caco-2 cells, as illustrated by increased TEER values and enhanced Caco-2 cell viability. Pre-treatment of Vero cell monolayers with all forms of encapsulated bLf followed by exposure to toxin B or bacterial supernatant induced a fall in mitochondrial enzyme activity.Conclusions
Our results are the first to suggest that alginate-bLf microparticles show protective effects against C. difficile toxin-mediated mucosal damage and impairment of barrier function in hIECs. The future potential of lactoferrin-loaded alginate microparticles in the treatment and prevention of CDI deserves further investigation in preclinical studies.