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.

To reliably infect a primate model for human immunodeficiency virus (HIV), ϳ10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ϳ4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.Cervicovaginal mucus (CVM) and semen from human immunodeficiency virus (HIV)-infected individuals contain cellfree and cell-associated HIV (8,25,26). Both forms of virions are plausible mediators of infection, and both, to be infectious, must penetrate the mucus barrier that coats and adheres to vaginal and penile epithelia during coitus. To the extent that mucus can limit the amount of virus that contacts the epithelium, the mucus layer can reduce the probability of infection. Leukocytes can migrate through neutral mucus (24) but are rapidly immobilized and then killed by mild acidity (pH Յ 6) (22); leukocytes do not survive in the acidic vagina. However, prior research has not revealed whether cell-free HIV can penetrate human CVM.To directly determine whether cell-free HIV can diffuse through CVM, we used a HIV virus-like particle (VLP) that was fluorescently labeled internally by incorporation of a green fluorescent protein (GFP)-Vpr fusion (5). For biosafety considerations, the derivative was replication defective and pseudotyped with X4-tropic HIV envelope. We mixed the labeled HIV at minimal dilution (ϳ3% [vol/vol]) into fresh, undiluted CVM obtained from donors with normal lactobacillus-dominated vaginal flora and observed the translational movements of hundreds of individual HIV virions in each sample using high-resolution multiple-particle tracking (15,29). CVM from women with healthy vaginal microflora is acidified to pH ϳ4 by lactic acid produced continuously by anaerobic metabolism of the lactobacilli (4, 21). However, during coitus, vaginal secretions are temporarily neutralized by the alkaline pH of semen (9, 30). In addition, women with bacterial vaginosis (BV), a condition that leads to a more neutral vaginal pH of ϳ5 to 6 (4), are at significantly increased risk of acquiring HIV infection (1, 28). Ther...

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Short-chain fatty acid (SCFA)-carbohydrate hybrid molecules that target both histone deacetylation and glycosylation pathways to achieve sugar-dependent activity against cancer cells are described in this article. Specifically, n-butyrate esters of N-acetyl-D-mannosamine (But4ManNAc, 1) induced apoptosis, whereas corresponding N-acetyl-D-glucosamine (But4GlcNAc, 2), D-mannose (But5Man, 3), or glycerol (tributryin, 4) derivatives only provided transient cell cycle arrest. Western blots, reporter gene assays, and cell cycle analysis established that n-butyrate, when delivered to cells via any carbohydrate scaffold, functioned as a histone deacetylase inhibitor (HDACi), upregulated p21WAF1/Cip1 expression, and inhibited proliferation. However, only 1, a compound that primed sialic acid biosynthesis and modulated the expression of a different set of genes compared to 3, ultimately killed the cells. These results demonstrate that the biological activity of butyrate can be tuned by sugars to improve its anticancer properties.

Recently, 24nm polymer nanoparticles were found to access a privileged non-degradative intracellular pathway that leads to perinuclear accumulation. Here, we report the intracellular dynamics of vesicles containing polymer nanoparticles within this non-degradative pathway, characterized by clathrin-and caveolae-independent endocytosis, as compared to endosomes originating from classical clathrin-mediated endocytosis. Similar to transport of acidic endosomes and lysosomes, the dynamic movements of non-degradative vesicles exhibit substantial heterogeneity, including caged diffusion and pearls-on-a-string trajectories, a reflection of microtubule-dependent active transport that leads to rapid accumulation near the cell nucleus. However, the ensemble-averaged intracellular transport rate of vesicles in the non-degradative pathway is 4-fold slower than that of the acidic vesicles of late endosomes and lysosomes, highlighted by a 3-fold smaller fraction of actively transported vesicles. The distinct intracellular dynamics further confirms that small nanoparticles are capable of entering cells via a distinct privileged pathway that does not lead to lysosomal processing. This non-degradative pathway may prove beneficial for the delivery of therapeutics and nucleic acids to the nucleus or nearby organelles.