Thermosensitive and mucoadhesive pluronic-hydroxypropylmethylcellulose hydrogel containing the mini-CD4 M48U1 is a promising efficient barrier against HIV diffusion through macaque cervicovaginal mucus.

To be efficient, vaginal microbicide hydrogels should form a barrier against viral infections and prevent virus spreading through mucus. Multiple particle tracking was used to quantify the mobility of 170-nm fluorescently labeled COOH-modified polystyrene particles (COOH-PS) into thermosensitive hydrogels composed of amphiphilic triblock copolymers with block compositions EOn-POm-EOn (where EO refers to ethylene oxide and PO to propylene oxide) containing mucoadhesive hydroxypropylmethylcellulose (HPMC). COOH-PS were used to mimic the size and the surface charge of HIV-1. Analysis of COOH-PS trajectories showed that particle mobility was decreased by Pluronic hydrogels in comparison with cynomolgus macaque cervicovaginal mucus and hydroxyethylcellulose hydrogel (HEC; 1.5% by weight [wt%]) used as negative controls. Formulation of the peptide mini-CD4 M48U1 used as an anti-HIV-1 molecule into a mixture of Pluronic F127 (20 wt%) and HPMC (1 wt%) did not affect its anti-HIV-1 activity in comparison with HEC hydrogel. The 50% inhibitory concentration (IC50) was 0.53 µg/ml (0.17 µM) for M48U1-HEC and 0.58 µg/ml (0.19 µM) for M48U1-F127-HPMC. The present work suggests that hydrogels composed of F127-HPMC (20/1 wt%, respectively) can be used to create an efficient barrier against particle diffusion in comparison to conventional HEC hydrogels.

Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: demonstration of diamagnetic chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI).

Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging ("theranostics"). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥7 mol% PEG diffused only ~10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7 mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3mol%-PEG liposomes. However, increasing PEG content to ~12 mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7 mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. FROM THE CLINICAL EDITOR: This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.

Pretreatment of human cervicovaginal mucus with pluronic F127 enhances nanoparticle penetration without compromising mucus barrier properties to herpes simplex virus.

Mucosal drug delivery nanotechnologies are limited by the mucus barrier that protects nearly all epithelial surfaces not covered with skin. Most polymeric nanoparticles, including polystyrene nanoparticles (PS), strongly adhere to mucus, thereby limiting penetration and facilitating rapid clearance from the body. Here, we demonstrate that PS rapidly penetrate human cervicovaginal mucus (CVM), if the CVM has been pretreated with sufficient concentrations of Pluronic F127. Importantly, the diffusion rate of large polyethylene glycol (PEG)-coated, nonmucoadhesive nanoparticles (PS-PEG) did not change in F127-pretreated CVM, implying that F127 did not significantly alter the native pore structure of CVM. Additionally, herpes simplex virus type 1 (HSV-1) remains adherent in F127-pretreated CVM, indicating that the presence of F127 did not reduce adhesive interactions between CVM and the virions. In contrast to treatment with a surfactant that has been approved for vaginal use as a spermicide (nonoxynol-9 or N9), there was no increase in inflammatory cytokine release in the vaginal tract of mice after daily application of 1% F127 for 1 week. Pluronic F127 pretreatment holds potential as a method to safely improve the distribution, retention, and efficacy of nanoparticle formulations without compromising CVM barrier properties to pathogens.

Nanoparticle-mediated drug delivery to treat infections in the female reproductive tract: evaluation of experimental systems and the potential for mathematical modeling.

A variety of drug-delivery platforms have been employed to deliver therapeutic agents across cervicovaginal mucus (CVM) and the vaginal mucosa, offering the capability to increase the longevity and retention of active agents to treat infections of the female reproductive tract (FRT). Nanoparticles (NPs) have been shown to improve retention, diffusion, and cell-specific targeting via specific surface modifications, relative to other delivery platforms. In particular, polymeric NPs represent a promising option that has shown improved distribution through the CVM. These NPs are typically fabricated from nontoxic, non-inflammatory, US Food and Drug Administration-approved polymers that improve biocompatibility. This review summarizes recent experimental studies that have evaluated NP transport in the FRT, and highlights research areas that more thoroughly and efficiently inform polymeric NP design, including mathematical modeling. An overview of the in vitro, ex vivo, and in vivo NP studies conducted to date - whereby transport parameters are determined, extrapolated, and validated - is presented first. The impact of different NP design features on transport through the FRT is summarized, and gaps that exist due to the limitations of iterative experimentation alone are identified. The potential of mathematical modeling to complement the characterization and evaluation of diffusion and transport of delivery vehicles and active agents through the CVM and mucosa is discussed. Lastly, potential advancements combining experimental and mathematical knowledge are suggested to inform next-generation NP designs, such that infections in the FRT may be more effectively treated.

Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions.

Cervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50 nM and 100 nM of siE6. Lipoplexes formulations induce 30-40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production.

Factors influencing nonoxynol-9 permeation and bioactivity in cervical mucus.

The effects of delivery gel pH and osmolarity on both the mass transport and 'biodiffusion' of the spermicide nonoxynol-9 (N9) in bovine cervical mucus were evaluated. Delivery gels were calcium chloride crosslinked alginate containing 3% N9, and were manufactured over a pH range of 3.4 to 5.9 and an osmolarity range of 300 to 900 mosmol. Mass transfer parameters (diffusion coefficients and total drug loading) were determined using a new UV spectrophotometric technique while biodiffusion (the diffusion distance into mucus at which sperm are killed) was assessed using the Double Ended Test. It was found that delivery gel pH had a significant effect on spermicidal efficacy of the alginate-N9 system; biodiffusion increased with decreasing pH. Actual N9 diffusion into mucus was found to be influenced by both the delivery gel pH and osmolarity. At high N9 concentration (near the gel/mucus interface), mass transport tended to decrease with decreasing pH at the highest osmolarity. At low concentration, mass transport tended to decrease with increasing osmolarity and decrease with increasing pH at the highest osmolarity. The difference between low and high concentration behavior can be attributed to N9 micelle formation. These findings are interpreted in the context of the design of intravaginal drug delivery vehicles for spermicides.

Comparison of the action of nonoxynol-9 and chlorhexidine on sperm.

The effect on sperm motility of nonoxynol-9 chlorhexidine diacetate was compared in semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml. When these compounds were allowed to diffuse into mucus, the subsequent survival of sperm in the mucus was different. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml chlorhexidine. When the compounds were mixed directly with the mucus before sperm penetration was attempted, chlorhexidine still immobilized sperm, but concentrations of nonoxynol-9 that would be spermicidal in semen had no effect in mucus.

PIP: This study compared the effect of nonoxynol-9 and chlorhexidine in sperm motility in both semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml, although the reduction of the proportion of motile sperm at lower concentrations was more marked for chlorhexidine. When these compounds were diffused into mucus, sperm survival showed different patterns. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml of chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml of nonoxynol-9. When the compounds were mixed directly with mucus before sperm penetration, chlorhexidine still immobilized sperm, but concentrations of nonoxynol-9 that would be spermicidal in semen had no effect. These findings suggest that nonoxynol-9 does not have access to the domains of the cervical mucus occupied by the swimming sperm; moreover, there is no evidence that this compound is able to diffuse into mucus. The nonoxynol-9 type of spermicide is probably active only in the vagina and sperm that enter the cervical mucus before being immobilized by the spermicide are protected from further action. In contrast, chlorhexidine may structurally alter the cervical mucus, preventing the entry of sperm, or may actively accumulate in mucus to spermicidal levels. The effectiveness of spermicidal vaginal contraception could be improved by extending the area of the female genital tract available to the contraceptive agent. Chlorhexidine provides such an extension.

Effects of a single contraceptive Silastic implant containing nomegestrol acetate on ovarian function and cervical mucus production during 2 years.

OBJECTIVE: To study the mechanism of action of Uniplant (South to South Corporation in Reproductive Health, Salvador, Brazil), a single Silastic capsule containing nomegestrol acetate (Lutenyl, Theramex, France) in women during 2 years. DESIGN: Comparison between the hormonal levels, follicular development, cervical mucus (CM) production, and endometrial thickness in the menstrual cycle before implant insertion and at 1, 6, 12, 18, and 24 months after implant insertion. PARTICIPANTS: A total of 15 women of reproductive age were enrolled for the 1st year of use. Twelve of these women continued for a 2nd year of Uniplant use. MAIN OUTCOME MEASURES: Hormonal plasma levels were measured in control cycles and at 1, 6, 12, 18, and 24 months of Uniplant use. Cervical mucus, follicular development, and endometrial thickness also were evaluated. RESULTS: In this study, Uniplant blocks ovulation in 86 percent of cycles studied. Disturbances in follicular growth were observed also. Cervical mucus was scanty and viscous in all women during this study. Endometrial thickness was <8 mm in all cycles studied. CONCLUSION: This study shows that Uniplant is a long-acting contraceptive that probably acts at the hypothalamic-pituitary levels, on the ovary, on CM production, and on the endometrium. These properties suggest the use of Uniplant as a contraceptive agent, especially if one considers the lack of androgenic and metabolic effects and the maintenance of periodic bleeding similar to menstruation.

PIP: A total of 15 healthy women volunteers were enrolled in this study. Their mean age was 23 +or- 1.2 years (range, 18-33 years), mean weight was 55.7 +or- 2.6 kg (range, 40-72 kg), and mean parity was 1.1 (range, 0-4). Venous blood samples were drawn every other day from day eight of the cycle until sonographic evidence of a 12-mm follicle, and then every day until sonographic evidence of follicular rupture and thereafter every other day until the next menstrual bleeding. The capsules were removed at the end of one year of Uniplant use, and a new capsule was inserted in 12 subjects. The blood samples for hormonal analyses were taken after 18 and 24 months of Uniplant use in the 12 women who continued in the study. Levels of luteinizing hormone (LH) were significantly lower than in the control cycles during the observation period (p .01, p .05). According to transvaginal sonography, four different patterns of follicular development were found: normal follicular growth and rupture, persistent follicle, follicular cysts, and no follicular growth. Follicular growth and rupture were observed in 20% of the treated cycles. Persistent follicles were present in approximately 15% of the treated cycles. Follicular cysts were observed in 29% of the cycles studied during 24 months of Uniplant use. All subjects had normal cervical cytology before starting treatment, after 12 months, and after 24 months of Uniplant use. The maximum cervical mucus score for pretreatment cycles was 12.8 +or- 0.4. Endometrial thickness was 8 mm in all cycles studied. 58% (7 of 12) of the women showed a normal menstrual cycle (26 to 32 days). 33% (4 of 12) of the women experienced one or two episodes of amenorrhea (90-134 days), whereas 8.3% of women (1 of 12) experienced episodes of spotting, six times in a period of 24 months of Uniplant use (10-30 days). Before Uniplant insertion, plasma concentration of sex hormone binding globulin was 72.3 nmol/L. After 24 months of Uniplant use, the concentration was 78.0 nmol/L.

Efficacy of nonhormonal vaginal contraceptives from a hydrogel delivery system.

This investigation describes the synthesis of a biodegradable hydrogel composed of a core surrounded by four concentric sheaths containing dextran, copolymers of polylactide and epsilon-caprolactone. The hydrogel was impregnated with iron (II) d-gluconate dihydrate, which causes complete spermiostasis due to lipid peroxidation, ascorbic acid to increase the viscosity of the cervical mucus and mixtures of polyamino and polycarboxylic acids to sustain vaginal pH close to 4.5. The combined effects of the agents in the daily eluates of the hydrogel were efficacious up to 16 days, within 30 s, as shown by sperm penetration tests. For in vivo studies, rabbits were chosen as the experimental model because they are easy to handle and the female is always in estrus. The anterior vagina of estrous female rabbits was instilled with the hydrogel, and then inseminated with the semen from a fertile male. Postinsemination flush from the female rabbits showed that all of the sperm were dead. These observations demonstrate the potential for the development of a biocompatible, nonhormonal, intravaginal contraceptive device.

Measurement and modulation of nonoxynol-9 diffusion and bioactivity against spermatozoa in human cervical mucus.

Assays of sperm penetration into cervical mucus, in the configuration of double-ended tests (DETs), are an accepted format for evaluating the efficacy of sperm-directed contraceptives in mucus. In order to distinguish relative contributions of compound permeation into, and compound bioactivity within, cervical mucus with respect to vanguard sperm penetration measured in DETs, direct measurements were made of concentration profiles of the spermicide nonoxynol-9 (N9) after diffusion into mucus-filled capillary tubes. N9 was dissolved in two different delivery solutions, deionized water and saline, in an attempt to exploit a Donnan-mediated swelling of mucus for enhanced delivery of the spermicide. Average diffusion coefficients, 7 and 5 x 10(-7) cm2/sec for N9-water and N9-saline, respectively, indicate that the diffusion of N9, a surfactant material, is governed by the size of the N9 micelle rather than the molecular size, in the concentration range typically found in commercial preparations Permeation of N9 into mucus was significantly greater for water versus saline as delivery solution, although the difference was slight. A more pronounced difference between the two treatments was found in DET results, due to an osmotic and/or pH activity of the delivery solution itself against sperm in mucus.

The activity of candidate virucidal agents, low pH and genital secretions against HIV-1 in vitro.

The effect of low pH, normally present in the female genital tract, on HIV viability was examined. HIV is more acid stable than previously reported with no substantial reduction in infectivity occurring until pH levels are reduced below 4.5. The virucidal activity of 3 topical spermicides and chlorhexidine was assessed in vitro using previously established and newly modified assay systems. None of the agents tested had a selectivity index (SI) greater than 5.2. Semen and cervical secretions were assessed for their ability to inhibit HIV-1. While no virucidal effect was found in the latter, seminal fluid was found to have significant activity against HIV-1 and a SI of approximately 50.

Development and characterization of bioadhesive vaginal films of sodium polystyrene sulfonate (PSS), a novel contraceptive antimicrobial agent.

PURPOSE: Polystyrene sulfonate (PSS) is a novel noncytotoxic antimicrobial contraceptive agent. A gel formulation of PSS was found safe for vaginal administration in phase I clinical trials. The purpose of the current study was to develop and evaluate novel bioadhesive vaginal film formulations of PSS. METHODS: PSS films were prepared by solvent evaporation and optimized for various physical, mechanical, and aesthetic properties. Further, films were evaluated for various biological activities and safety. RESULTS: Vaginal films containing 300 mg PSS per unit have been developed, using generally regarded as safe (GRAS) listed excipients. The films are colorless, transparent, thin, soft, and tough, dissolve rapidly in physiologic fluid to form a smooth, viscous and bioadhesive solution that could be retained in the vagina for prolonged intervals. Sperm function inhibition (hyaluronidase and cervical mucus penetration) and antimicrobial activities against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) by PSS films were found comparable to PSS. Also, films did not inhibit normal vaginal microflora (Lactobacillus) and were noncytotoxic as indicated by negligible sperm immobilization and cytotoxicity to host cell assays. CONCLUSIONS: Rapidly dissolving bioadhesive vaginal film formulation of PSS with desired physical, mechanical, aesthetic, and biological properties is a suitable candidate vaginal microbicide for prevention of sexually transmitted disease (STDs) and is ready for toxicological and clinical evaluation.

Some molecular features of human cervical mucus.

1) The time preservation effect of the enzymatic activities present in human cervical mucus has been studied. 2) In order to protect the enzymatic activities of the mucus from bacterial impurities, the enzymes studied were assayed in presence of NaN3. 3) The effect of an acid pH likes present in the vagina was also studied: a low pH exerts a marked inhibition on mucus enzymes. 4) the use of nonionic detergents for mucus solubilization does not produce a marked increase of the enzymatic activities studied. 5) A NADP-dependent malate dehydrogenase in peri- and post-ovulatory period has been identified. 6) The presence in mucus of at least four lactate dehydrogenase isoenzymes has been demonstrated and their absolute values determined.

[Study in vitro of the spermicidal action of the drug combination: nonylphenoxypolyethoxyethanol-dodecaethyleneglycol monolaurate-benzalkonium chloride]. / Studio in vitro sull'azione spermicida dell'associazione farmacologica nonilfenossipolietoessietanolo-dodecaetilenglicole monolaurato-benzalconio cloruro)

PIP: The spermicidal action of a new pharmacological association (nonilphenoxylpoliethoxyethanol, dodecaethilenglycol monolaurate, and benzalconium chloride) was investigated in vitro on the seminal liquid of 20 men, and on a mixture of seminal liquid and cervical mucus obtained from 9 women. Accurate spermiograms were done before and after the investigation. Results showed that the spermicidal action of the association is strong enough to be able to kill most spermatozoa, and that effectiveness depends on the strength of the dilution and on the time of application. It is also obvious that cervical mucus exercises a protective action in favor of spermatozoa. Conditions in vitro, however, are different from conditions in vivo, and therefore results must be considered for the time being, as only indicative. The clinical application of this pharmacological association should have all advantages and disadvantages of chemical spermicidal used vaginally.^ieng

Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives.

Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.