Improved oral therapeutic potential of nanoencapsulated cryptdin formulation against Salmonella infection

Abstract

An encapsulated system for cryptdin-2 (a Paneth cell antimicrobial peptide) was developed, with a view to help it sustain adverse gut conditions and to ensure its bioavailability on oral administration. The formulation was characterized on the basis of particle size, zeta potential and polydispersity index. Cryptdin-2 loaded nanoparticles of size 105 ± 7 nm, formulated by ionotropic gelation method using chitosan: tripolyphosphate (5:2), revealed 60% drug entrapment efficiency with 65% in vitro release in 4.5 h. Developed system was evaluated for its therapeutic application against Salmonella Typhimurium infection in mice, on the basis of survivability of animals, bacterial load in tissues, histo-architecture and oxidative damage markers. Infected mice when treated with the encapsulated peptide showed 83% survivability and approximately 2 log unit reductions in the bacterial load in the tissues versus 100% mortality observed with the free peptide. The encapsulated cryptdin-2 also achieved a decrease in the level of oxidants, particularly nitrite by 3.25 folds and increased the level of antioxidant catalase by 2 folds when compared to the levels exhibited by the free peptide. The bacteriological and biochemical alterations illustrated by encapsulated peptide co-related well with the histo-architectural studies. The study is a first pre-clinical report on the oral effectiveness of cryptdin-2 by its suitable encapsulation and has potential for future clinical applications.

Introduction

Salmonellosis continues to be one of the major public health issues worldwide (Pui et al., 2011). With the emergence of antibiotic resistance in Salmonella strains, the efforts to identify and exploit alternative antibacterial therapies against Salmonella infections are gaining pace. Scientific interest has now been directed towards various biotherapeutics agents, including peptides having antibacterial as well as immunomodulatory properties from eukaryotic origin (Bulet et al., 2004). In this context, Paneth cell peptides, known as cryptdins have been reported to exhibit microbicidal activity against several intestinal pathogens (Aley et al., 1994, Vaishnava et al., 2008, Preet and Rishi, 2010a, Ouellette, 2011, Preet et al., 2011).

Recently, the therapeutic potential of cryptdin-2 following subcutaneous route of administration against Salmonella infection has been documented by us (Preet and Rishi, 2010b, Preet et al., 2013, Rishi et al., 2011). However, oral therapy being user friendly is always a preferred route for drug administration, provided the major limitation of oral delivery i.e. the peptide degradation by harsh gut environment is avoided in addition to achieving the required bioavailability. Uptake of free peptide which is cationic and highly hydrophilic should in itself be a limitation to uptake across the intestinal mucosa. In order to overcome host barriers, thereby enhancing the in vivo performance of the peptide by assigning it a suitable covering, was presently perceived by us as a probable beneficial strategy. In recent years, encapsulation of antimicrobial drugs in micro/nano particulate systems has emerged as a pioneering and promising substitute that enhances therapeutic effectiveness and minimizes undesirable side effects of these agents, if any (Malmsten, 2013, Bysell et al., 2011). Therefore, nanoencapsulation may ensure close and intimate contact with the gut mucosa and allowing either a prolonged and continuous release of the peptide or uptake of intact nanoparticles containing the peptide. This is the first report on the efficiency of therapeutic effectiveness of cryptdin when administered orally having a suitable pharmaceutical covering against Salmonella infection.