Altering mucus rheology to "solidify" human mucus at the nanoscale

Abstract

The ability of mucus to function as a protective barrier at mucosal surfaces rests on its viscous and elastic properties, which are not well understood at length scales relevant to pathogens and ultrafine environmental particles. Here we report that fresh, undiluted human cervicovaginal mucus (CVM) transitions from an impermeable elastic barrier to non-adhesive objects sized 1 microm and larger to a highly permeable viscoelastic liquid to non-adhesive objects smaller than 500 nm in diameter. Addition of a nonionic detergent, present in vaginal gels, lubricants and condoms, caused CVM to behave as an impermeable elastic barrier to 200 and 500 nm particles, suggesting that the dissociation of hydrophobically-bundled mucin fibers created a finer elastic mucin mesh. Surprisingly, the macroscopic viscoelasticity, which is critical to proper mucus function, was unchanged. These findings provide important insight into the nanoscale structural and barrier properties of mucus, and how the penetration of foreign particles across mucus might be inhibited.

Figure 1. Nanoscale viscoelastic properties of fresh, undiluted human cervicovaginal mucus (CVM).

(A–D) Representative trajectories of 100 nm (A), 200 nm (B), 500 nm (C), and 1,000 nm (D) probe particles in CVM traced over 20 s. Particles exhibit an effective diffusivity within one s.e.m. of the ensemble mean. (E–H) Local elastic (G′, solid lines) and viscous (G″, dashed lines) moduli as a function of frequency for the same probe particles: 100 nm (E), 200 nm (F), 500 nm (G), and 1,000 nm (H).

Figure 2. Effect of nonoxynol-9 (N9) on the nanoscale viscoelastic properties of fresh human cervicovaginal mucus (CVM).

(A–C) Local elastic (G′) and viscous (G″) moduli as a function of frequency for 100 nm (A), 200 nm (B), and 500 nm (C) probe particles in N9-treated CVM. Representative trajectories of 200 nm (B inset) and 500 nm (C inset) probe particles, with an effective diffusivity within one s.e.m. of the ensemble average. (D) Phase angle (δ) at a frequency of 2π rad/s for probe particles in native or N9-treated CVM compared to bulk values (“B”) at the same frequency (mean±s.e.m.). The phase angle for a purely viscous fluid is 90°, while that for a purely elastic solid is 0°. (E) Dynamic viscosity (η″) at a frequency of 2π rad/s for probe particles in native or N9-treated CVM compared to bulk values (“B”) at the same frequency (mean±s.e.m.). The dashed line represents the viscosity of water. * denotes statistical significance (P<0.05).

Figure 3. Macrorheological characterization of fresh human cervicovaginal mucus (CVM) under dynamic oscillatory shear.

(A–D) Elastic modulus (G′) (A), viscous modulus (G″) (B), phase angle (δ) (C), and dynamic viscosity (η″) (D) for untreated (−N9), saline-treated (+Saline), or nonoxynol-9-treated (+N9) CVM.

Figure 4. Summary of the interpretation of results.

(A) The mesh structure of native human cervicovaginal mucus (CVM) consists of individual mucin fibers bundled together, leading to large mesh spacings. (B) The microrheology of CVM, quantified using different sized probe nanoparticles, suggests that CVM is largely a viscoelastic fluid at length scales 500 nm or below, whereas at length scales 1,000 nm or higher the mesh elements contribute to a markedly greater local elasticity characteristic of viscoelastic solids. (C) The macrorheology of CVM reflects contributions from entanglements as well as hydrophobic adhesive interactions between the mesh elements. (D) Treatment of CVM with nonoxynol-9 (N9) leads to unbundling of the mesh elements and significantly reduced mesh spacings, due to reduced hydrophobic interactions between mucin fibers. (E) The microrheology of N9-treated CVM becomes that of a viscoelastic solid at length scales down to 200 nm, but remains largely unperturbed at length scales ∼100 nm or below. (F) The effect of N9 cannot be probed by macrorheology, as the reduction in adhesive interactions by N9 is likely balanced by increased entanglements between mucin fibers.