The next development in the field of targeted delivery is cytoadhesins which are molecules (e.g. lectins) that bind directly to the epithelial cell surface instead of the mucus layer. Beyond receptor-mediated binding, lectins could trigger the active vesicular uptake of large molecules or small vehicles [176].
44
Figure 1.9: Polymeric structure of pGlcNAc [417]
Figure 1.10: Different types of liposomes [418]
(a) Originally, liposomes are vesicles with a lipid bilayer shell. A liposome can carry small (a few nm) hydrophobic functional molecules (red spheres) within the hydrophobic bilayer, and larger (several hundred nm) hydrophilic molecules (green star) in its inner cavity. (b) 'Stealth' liposomes are designed for drug-delivery and few polymer lipids are integrated into the lipid bilayer. Targeting peptides (blue rectangle) can also be introduced. (c) Liposome–DNA complexes (cationic) most often have a layered structure, with DNA (purple rods) orientated between the cationic membranes. (d) Kubitschke et al. [419] describe liposomes with a bilayer made of cavitands (vase-shaped molecules) with attached hydrophobic and hydrophilic chains. The cavitands can trap ångström-sized structures (yellow diamonds) in their hydrophobic cavities.
45
1.3.3 Nanostructures
More recently, technology based on nanosized structures has become relevant for food science. This is possible because targeting and controlled release delivery systems are linked to size and size distribution [420]: monodisperse populations perform better than polydisperse distributions in controllability of dose and release behaviour, drug encapsulation efficiency and biocompatibility with cells and tissues of the body [420-422]. Generally, nanosized particles have a higher binding capability and accumulation than larger particles at the target sites and trigger less immune response [421, 423].
Membrane emulsification is the preferable technique to produce nanoemulsions [416] as it allows the production of different types of emulsions, solid lipid microcapsules, polymer microspheres and ready microcapsules [424-427].
Prego et al. [428] made chitosan coated particles which adhere to a Caco-2 cell monolayer,after only one hour of incubation. However, the vehicles remained at the apical side. Applied to co- cultured Caco-2/HT29-M6 cells, the particles showed interactions with the cells, whereby nanocapsules were specifically located on the top of the goblet islets, demonstrating mucoadhesive character [429]. Unfortunately, the ability of chitosan to enhance absorption is reduced in mucus-covered cultures [430]. Further, acid-soluble chitosan is prone to precipitation upon reaching the neutral pH in the intestinal region [431]. Precipitation results in reduced swelling, a prerequisite for muco-adhesion [432], which was found to even cause detachment of previously mucin-bound chitosan [433].
A comparison of three nanoparticle constructs with different surface charges and hydrophobicity showed that mucus on IEC in culture has crucial influence on adhesion [434]. Polystyrene (hydrophobic) adheres better to Caco-2 than to HT29-MTX-E12 cultures, whereas chitosan (mucoadhesive) shows inverse behaviour. Poly-lactic acid-poly-ethylene glycol (negative surface charge, hydrophilic) did not adhere well to either cell-type. Reportedly there are indications of stronger interactions of positively charged nanoparticles with bio-membranes
46
than negative ones. However, positive surface charge seems to increase the transepithelial electric resistance value [435].
1.3.4 Liposomes
Liposomes are spherical structures of one or more phospholipid bilayers enclosing an aqueous core [436, 437]. They can be used for the entrapment and controlled release of hydrophilic and hydrophobic drugs or nutraceuticals or DNA (Figure 1.10) [438]. In addition, other molecules like antibodies or binding proteins can be anchored into liposomes or capsule surfaces, e.g. [439, 440]. However, none of these authors tested liposome targeting of any of the described human intestinal surface layers (Figure 1.6). More recently, liposomes have been appended with surface molecules, for example to allow “stealth” behaviour. Assembling the liposomal surface layer from vase shaped molecules allowed the delivery of functional molecules (Figure 1.10) [418, 419].
In their native state, liposomes are rapidly degraded by bile salts and other GIT secretions, thus they are not suitable for oral delivery. This could be avoided by using polymerised liposomes [147]. Further coating with lectins could facilitate passive targeting of liposomes [441-444].
1.3.5 Anti-adhesive molecules
Molecules that inhibit bacterial adhesion can be considered relevant for the group of intestinal adhesins. In order to prevent bacteria from binding to the GIT surface they must bind either to the bacteria or the GIT surface. In the first case anti-adhesive molecules have the potential to interact with the bacterial biofilm in the human GIT, in the latter the anti-adhesins might be retained by the mucin layer or the epithelial cells themselves.
1.3.5.1 Milk components
Data suggest that human milk oligosaccharides (HMOs) from milk or colostrum are exceptional anti-adhesives for diarrhoeal pathogens [24-29]. In addition, porcine milk contains lipopolysaccharide-binding components: LF, soluble CD14, serum amyloid A [30], αS1-casein, β-casein and κ-casein [31] which might adhere to Gram-negative bacteria [32]. Thus many free
47
oligosaccharides from (human) milk as well as glycoproteins are considered to be soluble receptor analogues of IEC surface carbohydrates [445]; e.g. in vitro attachment to IEC lines of
enteropathogenic E. coli (EPEC) can be inhibited by the oligosaccharide fraction of human
milk, mainly due to fucosyloligosaccharides [33-35], and also by glycosylated proteins like LF or free secretory component. LF may contribute to defence against facultative intracellular bacteria by binding both, target cell membrane glycosaminoglycans and bacterial invasins [446, 447]. Finally, the serine protease activity of LF is considered to inhibit the growth of bacteria, e.g. enteropathogenic E. coli, by degrading colonisation proteins [447, 448]; reviewed by Ward
et al. [449]. Lactoferricin, a cationic peptide generated by the pepsin digestion of LF, has more potent bactericidal activity than the native protein [447, 450].
1.3.5.2 Glycosides
Sialyloligosaccharides from egg yolk have been shown to inhibit Staphylococcus enteritidis (S. enteritidis) adherence to Caco-2 cells, presumably due to high density of a lipoprotein fraction
that reduces adherence. Those from the water soluble fraction of delipidated egg yolk act as glycomimetics of GM1-oligosaccharide and inhibit toxin adherence [451, 452]. Mannooligosaccharides are a group of oligosaccharides with potential anti-adhesive activity: α- linked mannose (Man) residues are known to inhibit the adhesion of many enterobacterial species, including Salmonella and E. coli [453, 454].
Cranberry extract appears to contain a multitude of anti-infection and anti-adhesive substances [37-39]. The high concentration of fructose inhibits in vitro type 1 fimbriae-mediated E. coli
adhesion [455]. Proanthocyanidins (flavonoid or condensed tannin) and other high molecular weight compounds were shown to inhibit adherence of uropathogenic E. coli [456, 457]. These
authors suggest that the cranberry components act as receptor analogues.
Also pectic type and other water soluble oligosaccharides were suggested to have anti- adherence activity [96, 458, 459]. A high-molecular weight extract from tea reduced adherence of Helicobacter pylori to a human gastric epithelial cell line and Staphylococcus aureus (S.
48
aureus) to fibroblast epithelial cell line. An aqueous extract from carrots blocked
enteropathogenic E. coli binding to HEp-2 and human mucosal cells [96] with an acidic
oligosaccharide containing trigalacturonic acid as the active substance.