Diffusion of Macromolecules and Virus-Like Particles in Human Cervical Mucus

Abstract

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus

“…The most surprising findings were: (i) larger polymeric nanoparticles (up to 500 nm) with a dense polyethylene-glycol coating can diffuse through CV mucus with rates up to onefourth as fast as they would in pure water; and (ii) 100-nm particles moved much more slowly through CV mucus than either 200-or 500-nm particles. The faster transport of 200-and 500-nm particles, regardless of surface chemistry (COOH or PEG), not only is contrary to the expectation that smaller particles should move faster in mucus (17,18,27) but also directly opposes the earlier estimates of CV mucus mesh spacing (27,28). Greater steric hindrance from the mucin fiber network and elevated friction forces was expected, a priori, to result in slower transport for larger particles in CV mucus.…”

“…The mucus barrier has been cited as a critical bottleneck in the treatment of a variety of diseases (17,24,(31)(32)(33)(34), and it has been widely suggested that nanoparticles are unable to efficiently traverse mucus layers (35)(36)(37)(38)(39), including CV mucus (27). For applications in CV diseases, the need for improved particle transport is further underscored by: (i) the previous observation that polystyrene beads firmly adhere to mucin fibers in human CV secretions, rendering them completely immobile (27); (ii) there is 100-to 1,000-fold reduced D eff for herpes simplex virus (d ϭ 180 nm) in CV mucus compared with water (27); and (iii) there are existing estimates of CV mucus mesh pore size of 10 to at most 200 nm from fluorescence recovery after photobleaching (FRAP) and most electron microscopy studies (27,28). The prevalent dogma in the design of nanoparticle therapeutics targeted to mucosal epithelia is that large nanoparticles, preferred for higher drugencapsulation efficiency and favorable drug-release kinetics, are not capable of crossing mucosal barriers.…”

“…Other mucus constituents, such as lipids, salts, macromolecules, cellular debris, and water, work together with mucins to form a nanoscopically heterogeneous environment for nanoparticle transport, where the shear-dependent bulk viscosity is typically 100-10,000 times more viscous than water (24). Small viruses up to 55 nm have been shown to diffuse in CV mucus as rapidly as in water; however, a larger virus, 180-nm herpes simplex virus, was slowed 100-to 1,000-fold by CV mucus compared with water, suggesting that the mucus mesh spacing is Ϸ20-200 nm (27,28). It was also previously reported that polystyrene particles (59-1,000 nm) adhered tightly to cervical mucus, rendering them completely immobile (27).…”

“…Small viruses up to 55 nm have been shown to diffuse in CV mucus as rapidly as in water; however, a larger virus, 180-nm herpes simplex virus, was slowed 100-to 1,000-fold by CV mucus compared with water, suggesting that the mucus mesh spacing is Ϸ20-200 nm (27,28). It was also previously reported that polystyrene particles (59-1,000 nm) adhered tightly to cervical mucus, rendering them completely immobile (27). These observations have suggested that the transport of synthetic polymer nanoparticles, especially those larger than Ϸ59 nm, was unlikely to occur efficiently enough to allow access of sustained release particles to underlying epithelium in human mucuscovered tissues.…”

An exploration of the microrheological environment around the distal ileal villi and proximal colonic mucosa of the possum ( Trichosurus vulpecula )

“…These beads were chosen on the basis that their movements would be relatively restricted when they were located in mucins as they would adhere to hydrophobic regions of mucin proteins [37]. The size of beads was chosen to exceed the mean reported distance of around 100 nm between component strands of mucin [38] so as to prevent the sampling voids between adjacent strands of the mucin matrix [20]. Indeed, quantitative microrheology in such systems requires that the microbead size should be in excess of that of the polymer mesh.…”