Richard A. Cone

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus

Nanoparticles larger than the reported mesh-pore size range (10–200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of drugs. We used high-speed multiple-particle tracking to quantify transport rates of individual polymeric particles of various sizes and surface chemistries in samples of fresh human cervicovaginal mucus. Both the mucin concentration and viscoelastic properties of these cervicovaginal samples are similar to those in many other human mucus secretions. Unexpectedly, we found that large nanoparticles, 500 and 200 nm in diameter, if coated with polyethylene glycol, diffused through mucus with an effective diffusion coefficient (Deff) only 4- and 6-fold lower than that for the same particles in water (at time scale τ = 1 s). In contrast, for smaller but otherwise identical 100-nm coated particles, Deff was 200-fold lower in mucus than in water. For uncoated particles 100–500 nm in diameter, Deff was 2,400- to 40,000-fold lower in mucus than in water. Much larger fractions of the 100-nm particles were immobilized or otherwise hindered by mucus than the large 200- to 500-nm particles. Thus, in contrast to the prevailing belief, these results demonstrate that large nanoparticles, if properly coated, can rapidly penetrate physiological human mucus, and they offer the prospect that large nanoparticles can be used for mucosal drug delivery.

Addressing the PEG Mucoadhesivity Paradox to Engineer Nanoparticles that “Slip” through the Human Mucus Barrier

Mucus linings serve as the bodys first line of defense at exposed surfaces of the eye and respiratory, gastrointestinal, and cervicovaginal tracts. The high viscoelasticity and adhesivity of mucus traps and limits exposure to foreign pathogens,[1,2] toxins,[3] and environmental ultrafine particles,[1,4] which are all typically removed by normal mucus clearance mechanisms. Numerous studies have demonstrated that human mucus also strongly immobilizes conventional synthetic nanoparticles,[5-7] and therefore represents a hurdle for localized drug and gene delivery at mucosal surfaces, such as aerosol-based gene carriers for cystic fibrosis gene therapy.[8] To increase the bioavailability of cargo therapeutics, it is important that carrier particles rapidly penetrate mucus to avoid being shed.

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

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.

Antibody diffusion in human cervical mucus.

The mucosal immune system actively transports large quantities of antibodies into all mucus secretions, and these secreted antibodies help prevent infectious entry of many pathogens. Mucus is generally thought to protect epithelial cells by forming a diffusional barrier through which only small molecules can pass. However, electron microscopy indicates that the pore size in mucus is approximately 100 nm, which suggests that antibodies as well as other large molecules might also diffuse through mucus. We measured the diffusion coefficients for antibodies and other proteins within human midcycle cervical mucus using two techniques: fluorescence imaging of concentration profiles and fluorescence photobleaching recovery. The two techniques are complementary, since the rates of diffusion are observed over millimeter distances with fluorescence imaging of concentration profiles and micron distances with fluorescence photobleaching recovery. Both methods yielded essentially the same diffusion coefficients. In contrast to previous reports indicating mucus significantly impedes diffusion of small molecules, antibody diffusion in mucus was relatively unimpeded. In our observations IgG, IgG fragments, IgA, and IgM diffused almost as rapidly in cervical mucus as in water (1.0 > Dmucus/Dwater > 0.7). Simple models for diffusion through water-filled pores suggest that the hydrodynamic pore size for cervical mucus is approximately 100 nm, smaller than the approximately 1000 nm pore size of a collagen gel (at 1 mg/ml) and larger than the approximately 10 nm pore size of gelatin (at 100 mg/ml). This estimated pore size is consistent both with electron micrographs and geometric models of interfiber spacing. Based on these results, we predict that particles as large as viruses can diffuse rapidly through human midcycle cervical mucus, provided the particle forms no adhesive interactions with mucus glycoproteins.

A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes.

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti–herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.

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

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.

Human Immunodeficiency Virus Type 1 Is Trapped by Acidic but Not by Neutralized Human Cervicovaginal Mucus

ABSTRACT To reliably infect a primate model for human immunodeficiency virus (HIV), ∼10,000-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 ∼4 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.

Mucus-Penetrating Nanoparticles for Vaginal Drug Delivery Protect Against Herpes Simplex Virus

Mucus-penetrating particles improve drug delivery to the mucosal surface of the mouse vagina and deliver acyclovir for enhanced protection against vaginal herpes infection in mice. Thick and Sticky For most, mucus is an unfortunate, never-ending by-product of a summer cold. For those in nanomedicine, however, mucus represents a formidable barrier to delivering drugs to various tissues, including the sinuses and the vagina. Here, Ensign and colleagues have devised a nanoparticle that is capable of penetrating the thick mucus layer standing between drug and tissue of interest. To develop a drug delivery vehicle that could not only move through mucus, but also afford sustained release over time, Ensign et al. coated polystyrene or biodegradable poly(lactic-co-glycolic acid) nanoparticles with a low–molecular weight polymer commonly known as PEG. These mucus-penetrating particles (MPPs) moved quickly through mouse cervicovaginal mucus (CVM) when delivered in hypotonic solution, allowing them to penetrate deep into the vaginal folds within minutes and to remain there for 24 hours. Conversely, conventional, uncoated particles were stuck in the thick mucus layer, unable to reach the tissue below. Both particle types displayed the same behavior in human CVM ex vivo. Ensign et al. showed that these particles could protect against vaginal transmission of herpes simplex virus (HSV). Mice were administered either MPPs laden with a modified form of the drug acyclovir—which is used in humans to treat HSV outbreaks—or a soluble form of the drug, before challenge with the virus. Of the mice that received the MPPs, only 47% of the mice were infected with HSV, whereas 84% of the controls were infected after receiving soluble acyclovir at the same concentration. Although more tests will be needed to show protection against herpes in situations that more closely mimic the human vagina, these MPPs may be the key to safe and effective drug delivery to prevent and treat sexually transmitted infections. Incomplete coverage and short duration of action limit the effectiveness of vaginally administered drugs, including microbicides, for preventing sexually transmitted infections. We investigated vaginal distribution, retention, and safety of nanoparticles with surfaces modified to enhance transport through mucus. We show that mucus-penetrating particles (MPPs) provide uniform distribution over the vaginal epithelium, whereas conventional nanoparticles (CPs) that are mucoadhesive are aggregated by mouse vaginal mucus, leading to poor distribution. Moreover, when delivered hypotonically, MPPs were transported advectively (versus diffusively) through mucus deep into vaginal folds (rugae) within minutes. By penetrating into the deepest mucus layers, more MPPs were retained in the vaginal tract after 6 hours compared to CPs. After 24 hours, when delivered in a conventional vaginal gel, patches of a model drug remained on the vaginal epithelium, whereas the epithelium was coated with drug delivered by MPPs. We then developed MPPs composed of acyclovir monophosphate (ACVp). When administered before vaginal herpes simplex virus 2 challenge, ACVp-MPPs protected 53% of mice compared to only 16% protected by soluble drug. Overall, MPPs improved vaginal drug distribution and retention, provided more effective protection against vaginal viral challenge than soluble drug, and were nontoxic when administered daily for 1 week.

IN VAGINAL FLUID, BACTERIA ASSOCIATED WITH BACTERIAL VAGINOSIS CAN BE SUPPRESSED WITH LACTIC ACID BUT NOT HYDROGEN PEROXIDE

BackgroundHydrogen peroxide (H2O2) produced by vaginal lactobacilli is generally believed to protect against bacteria associated with bacterial vaginosis (BV), and strains of lactobacilli that can produce H2O2 are being developed as vaginal probiotics. However, evidence that led to this belief was based in part on non-physiological conditions, antioxidant-free aerobic conditions selected to maximize both production and microbicidal activity of H2O2. Here we used conditions more like those in vivo to compare the effects of physiologically plausible concentrations of H2O2 and lactic acid on a broad range of BV-associated bacteria and vaginal lactobacilli.MethodsAnaerobic cultures of seventeen species of BV-associated bacteria and four species of vaginal lactobacilli were exposed to H2O2, lactic acid, or acetic acid at pH 7.0 and pH 4.5. After two hours, the remaining viable bacteria were enumerated by growth on agar media plates. The effect of vaginal fluid (VF) on the microbicidal activities of H2O2 and lactic acid was also measured.ResultsPhysiological concentrations of H2O2 (< 100 μM) failed to inactivate any of the BV-associated bacteria tested, even in the presence of human myeloperoxidase (MPO) that increases the microbicidal activity of H2O2. At 10 mM, H2O2 inactivated all four species of vaginal lactobacilli but only one of seventeen species of BV-associated bacteria. Moreover, the addition of just 1% vaginal fluid (VF) blocked the microbicidal activity of 1 M H2O2. In contrast, lactic acid at physiological concentrations (55-111 mM) and pH (4.5) inactivated all the BV-associated bacteria tested, and had no detectable effect on the vaginal lactobacilli. Also, the addition of 10% VF did not block the microbicidal activity of lactic acid.ConclusionsUnder optimal, anaerobic growth conditions, physiological concentrations of lactic acid inactivated BV-associated bacteria without affecting vaginal lactobacilli, whereas physiological concentrations of H2O2 produced no detectable inactivation of either BV-associated bacteria or vaginal lactobacilli. Moreover, at very high concentrations, H2O2 was more toxic to vaginal lactobacilli than to BV-associated bacteria. On the basis of these in vitro observations, we conclude that lactic acid, not H2O2, is likely to suppress BV-associated bacteria in vivo.

Vaginal pH and Microbicidal Lactic Acid When Lactobacilli Dominate the Microbiota

Lactic acid at sufficiently acidic pH is a potent microbicide, and lactic acid produced by vaginal lactobacilli may help protect against reproductive tract infections. However, previous observations likely underestimated healthy vaginal acidity and total lactate concentration since they failed to exclude women without a lactobacillus-dominated vaginal microbiota, and also did not account for the high carbon dioxide, low oxygen environment of the vagina. Fifty-six women with low (0-3) Nugent scores (indicating a lactobacillus-dominated vaginal microbiota) and no symptoms of reproductive tract disease or infection, provided a total of 64 cervicovaginal fluid samples using a collection method that avoided the need for sample dilution and rigorously minimized aerobic exposure. The pH of samples was measured by microelectrode immediately after collection and under a physiological vaginal concentration of CO2. Commercial enzymatic assays of total lactate and total acetate concentrations were validated for use in CVF, and compared to the more usual HPLC method. The average pH of the CVF samples was 3.5 ± 0.3 (mean ± SD), range 2.8-4.2, and the average total lactate was 1.0% ± 0.2% w/v; this is a five-fold higher average hydrogen ion concentration (lower pH) and a fivefold higher total lactate concentration than in the prior literature. The microbicidal form of lactic acid (protonated lactic acid) was therefore eleven-fold more concentrated, and a markedly more potent microbicide, than indicated by prior research. This suggests that when lactobacilli dominate the vaginal microbiota, women have significantly more lactic acid-mediated protection against infections than currently believed. Our results invite further evaluations of the prophylactic and therapeutic actions of vaginal lactic acid, whether provided in situ by endogenous lactobacilli, by probiotic lactobacilli, or by products that reinforce vaginal lactic acid.