Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses

Abstract

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 +/- 70 nm; range approximately 50-1800 nm). This mesh structure is substantially more open than the 15-100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 +/- 50 nm. This suggests hydrophobic interactions between lipid-coated "naked" protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin "cables" at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

Fig. 1.

Transport of different-sized PEG-coated particles in minimally perturbed human CVM. (A) Ensemble-averaged geometric mean squared displacements (〈MSD〉) as a function of time scale. Error bars are presented as standard error of the mean (s.e.m). (B) Distribution of effective pore sizes, with average pore size 340 ± 70 nm (s.e.m.). Data represent the ensemble average of at least three independent experiments, with n≥120 particles for each experiment.

Fig. 2.

Transport of different-sized PEG-coated particles in detergent-treated human CVM. (A) Ensemble-averaged geometric mean squared displacements (〈MSD〉) as a function of time scale. Error bars are presented as s.e.m. (B) Distribution of effective pore sizes, with average pore size 130 ± 50 nm (s.e.m.). Data represent the ensemble average of at least three independent experiments, with n≥120 particles for each experiment.

Fig. 3.

Transport of HSV (diameter 180 nm) in minimally perturbed human cervicovaginal mucus compared to 200-nm non-mucoadhesive, PEG-coated particles. (A) Representative trajectories of HSV and 200-nm PEG-coated particles, with effective diffusivities within one s.e.m. of their ensemble average. (B) Ensemble-averaged geometric mean squared displacements (〈MSD〉) as a function of time scale. (C) Ratios of the ensemble average diffusion coefficients in mucus (D_m) compared to in water (D_w). Data represent three independent experiments for HSV, with n≥100 virus particles tracked for each experiment. The asterisk (*) indicates statistical significance (P < 0.005). Error bars are presented as s.e.m.