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Abstract Vaginal drug delivery has been shown to be a promising strategy for the prevention of sexually transmitted infections. Therapy delivered at the site of infection has many advantages including improved therapeutic efficacy, reduction in systemic toxicity, and reduced potential for development of drug resistance. We developed a “smart” combination intravaginal ring (IVR) that will (1) provide continuous release of hydroxychloroquine (HCQ) to induce T cell immune quiescence as the first-line of defense and (2) release nanoparticles containing anti-CCR5 siRNA only during sexual intercourse when triggered by the presence of seminal fluid as the second-line of defense. The IVR was capable of releasing HCQ over 25 days with a mean daily release of 31.17 ± 3.06 µg/mL. In the presence of vaginal fluid simulant plus seminal fluid simulant, over 12 × more nanoparticles (5.12 ± 0.9 mg) were released over a 4-h period in comparison to IVR segments that were incubated in the presence of vaginal fluid simulant alone (0.42 ± 0.19 mg). Anti-CCR5 siRNA nanoparticles were able to knockdown 83 ± 5.1% of CCR5 gene expression in vitro in the CD4+ T cell line Sup-T1. The IVR system also demonstrated to be non-cytotoxic to VK2/E6E7 vaginal epithelial cells. Graphical abstract

To achieve a pH-responsive switchable on-demand release of nanoparticles (NPs) from intravaginal rings (IVR), a new pH-sensitive polyurethane (PU) bearing dimethylolpropionic acid (PEG-DMPA-HDI-PG) was synthesized to encapsulate NPs as a physically cross-linked hydrogel within a segmented reservoir-IVR. A new PEGylated polyaspartic acid-based copolymer conjugated with the fluorescent dye Orange II (PASP-PEG-Ph-Orange) was synthesized to self-assemble in aqueous solution into NPs (251–283 nm) for the release study. Chemical structures of the PEG-DMPA-HDI-PG and PASP-PEG-Ph-Orange were confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and1H nuclear magnetic resonance (1H NMR) spectroscopy. PASP-PEG-Ph-Orange NPs showed the highest fluorescent emission at 570 nm for tracking, and PEG-DMPA-HDI-PG became a pH-responsive supramolecular hydrogel in distilled water at 20 wt %. PASP-PEG-Ph-Orange NPs were blended with the PEG-DMPA-HDI-PG hydrogel to form an inclusion complex and then ...

Efforts in developing an effective vaccine for human immunodeficiency virus (HIV) has been challenging as HIV strains are highly variable and exhibit extraordinary mutability. Despite condom usage and pre-exposure prophylaxis as excellent prevention strategies, lack of accessibility in some developing countries and low adherence due to sociocultural factors continue to act as barriers in reducing the HIV epidemic. Microbicides are topical therapies developed to prevent HIV and other sexually transmitted infections during intercourse. Microbicides applied vaginally or rectally are intended to prevent HIV infection at the site of transmission by either inhibiting its entry into immune cells or prevent viral replication. This review will summarize some of the current state-of-the-art microbicide formulations that are in preclinical and clinical stages of development and discuss some of the challenges associated with microbicide development.

Evidence suggests that women who are naturally resistant to HIV infection exhibit low baseline immune activation at the female genital tract (FGT). This "immune quiescent" state is associated with lower expression of T-cell activation markers, reduced levels of gene transcription and pro-inflammatory cytokine or chemokine production involved in HIV infection while maintaining an intact immune response against pathogens. Therefore, if this unique immune quiescent state can be pharmacologically induced locally, it will provide an excellent women-oriented strategy against HIV infection To our knowledge, this is the first research article evaluating in vivo, an innovative trackable implant that can provide controlled delivery of hydroxychloroquine (HCQ) to successfully attenuate vaginal T lymphocyte activation and inflammation in a rabbit model as a potential strategy to induce an "immune quiescent" state within the FGT for the prevention of HIV infection. This biocompatible implant can deliver HCQ above therapeutic concentrations in a controlled manner, reduce submucosal immune cell recruitment, improve mucosal epithelium integrity, decrease protein and gene expression of T-cell activation markers, and attenuate the induction of key pro-inflammatory mediators. Our results suggest that microbicides designed to maintain a low level of immune activation at the FGT may offer a promising new strategy for reducing HIV infection.

C. trachomatis is the most common sexually transmitted bacterial infection in the world. Although the infection can be easily controlled by the use of antibiotics, several reports of clinical isolates that are resistant to antibiotics have prompted us to search for alternative strategies to manage this disease. In this paper, we developed a nanoparticle formulation (PDGFR-β siRNA-PEI-PLGA-PEG NP) that can simultaneously induce autophagy in human cells and knock down PDGFR-β gene expression, an important surface binding protein for C. trachomatis, as a strategy to reduce vaginal infection of C. trachomatis. PDGFR-β siRNA-PEI-PLGA-PEG NP significantly induced autophagy in human vaginal epithelial cells (VK2/E6E7) 48 hr post treatment by improving autophagic degradation activity without causing inflammation, apoptosis or any decrease in cell viability. Beclin-1, VPS34 (markers for initiation stage of autophagy), UVRAG, TECPR-1 (markers for degradation stage of autophagy) were found to be significantly upregulated after treatment with PDGFR-β siRNA-PEI-PLGA-PEG NP. Furthermore, PDGFR-β siRNA-PEI-PLGA-PEG NP decreased PDGFR-β mRNA expression by 50% and protein expression by 43% in VK2/E6E7 cells 48 hr post treatment. Treatment of cells with PDGFR-β siRNA-PEI-PLGA-PEG NP significantly decreased the intracellular C. trachomatis and extracellular release of C. trachomatis by approximately 65% and 67%, respectively, in vitro through augmenting autophagic degradation pathways and reducing bacterial binding simultaneously.

The objective of this study was to develop and characterize a novel intravaginal membrane platform for pH-triggered release of nanoparticles (NPs), which is essential for efficient intravaginal delivery of certain effective but acid-labile therapeutic agents for sexually transmitted infections, such as small interfering RNA (siRNA). A pH-responsive polyurethane (PU) was electrospun into a porous nanofibrous membrane. The diameters of the fibers, as well as the thickness and pore sizes of the membrane under dry and wet conditions (pH 4.5 and 7.0), were determined from scanning electron microscopy (SEM) micrographs. pH-dependent zeta-potential (ζ) of the membrane was evaluated using a SurPASS electrokinetic analyzer. Visiblex™ color-dyed polystyrene NPs (PSNs, 200 nm, COOH) and CCR5 siRNA-encapsulated solid lipid NPs (SLNs) were used for in vitro NP release studies in a vaginal fluid simulant (VFS) at pH 4.5 (normal physiological vaginal pH) and 7.0 (vaginal pH neutralization by semen). During 24 h of incubation in VFS, close-to-zero PSNs (2 ± 1%) and 28 ± 4% SLNs were released through the PU membrane at pH 4.5, whereas the release of PSNs and SLNs significantly increased to 60 ± 6% and 59 ± 8% at pH 7.0, respectively. The pH-responsive release of NPs hinged on the electrostatic interaction between the pH-responsive membrane and the anionic NPs, and the change in pH-responsive morphology of the membrane. In vitro biocompatibility studies of the membrane showed no significant cytotoxicity to VK2/E6E7 human epithelial cells and Sup-T1 human T-cells and no significant changes in the expression of pro-inflammatory cytokines (IL-6, IL-8, and IL-1β). Overall, the porous pH-responsive PU membrane demonstrated its potential in serving as a “window” membrane in reservoir-type intravaginal rings (IVRs) for pH-responsive intravaginal release of NPs. Statement of Significance Stimuli-responsive intravaginal nanoparticle release is achieved for the first time through a new electrospun pH-responsive polyurethane (PU) semi-permeable membrane, which can serve as a “window” membrane in the reservoir-type IVR for the prevention of human immunodeficiency virus (HIV) transmission. Almost no release of nanoparticles was observed at normal pH in the female genital tract (in vaginal fluid simulant [VFS], at pH 4.5); however, a continuous release of NPs was observed at elevated pH in the female genital tract (in VFS, at pH 7.0). This pH-responsive intravaginal release can reduce side effect and drug resistance by avoiding unnecessary exposure. The PU semi-permeable membrane demonstrated potential use as biomaterials for “smart” intravaginal nanoparticle release and has great potential to protect women from HIV.

In this study, we investigated the viscoelastic and mechanical behaviour of polyvinyl alcohol films formulated along with carrageenan, plasticizing agents (polyethylene glycol and glycerol), and when loaded with nanoparticles as a model for potential applications as microbicides. The storage modulus, loss modulus and glass transition temperature were determined using a dynamic mechanical analyzer. Films fabricated from 2% to 5% polyvinyl alcohol containing 3 mg or 5 mg of fluorescently labeled nanoparticles were evaluated. The storage modulus and loss modulus values of blank films were shown to be higher than the nanoparticle-loaded films. Glass transition temperature determined using the storage modulus, and loss modulus was between 40–50℃ and 35–40℃, respectively. The tensile properties evaluated showed that 2% polyvinyl alcohol films were more elastic but less resistant to breaking compared to 5% polyvinyl alcohol films (2% films break around 1 N load and 5% films break around 7 N load). To our knowledge, this is the first study to evaluate the influence of nanoparticle and film composition on the physico-mechanical properties of polymeric films for vaginal drug delivery.

New amphiphilic PEGylated poly(aspartic acid) graft copolymer (PASP-PEG-Ph) was synthesized as a nanocarrier for intravaginal drug delivery of poorly water-soluble drugs. PASP-PEG-Ph self-assembled into negatively charged spherically shaped nanoparticles in the presence of pH 4.5 and pH 7.0 vaginal fluid simulants with a diameter of approximately 200 nm as evidenced by Zeta-potentiometer, scanning electron microscope (SEM), dynamic light scattering (DLS) analysis. A significant number of stable NPs could be maintained at pH 4.5, 37 °C for 13 days. The PASP-PEG-Ph NP showed no significant cytotoxicity toward the T-cell line SupT1 and human vaginal epithelial cell line Vk2/E6E7 up to 1 mg/mL. The highest encapsulation efficiency of the model drug coumarin 6 (C6) by PASP-PEG-Ph was 92.0 ± 5.7%. The sustained release profile of the encapsulated C6 was demonstrated by an in vitro release study. An in vitro cellular uptake study revealed strong cellular uptake of the C6 loaded NP by SupT1 cells within 2 h.

The use of polymeric devices for controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides, when topically applied vaginally, may be cytotoxic to vaginal epithelial cells and the protective microflora present within the female genital tract. In this study, we evaluated the impact of hydroxychloroquine (HCQ)-loaded, reservoir-type, polyurethane intravaginal rings (IVRs) on the growth of Lactobacillus crispatus and Lactobacillus jensenii and on the viability of vaginal and ectocervical epithelial cells. The IVRs were fabricated using hot-melt injection molding and were capable of providing controlled release of HCQ for 24 days, with mean daily release rates of 17.01 ± 3.6 μg/ml in sodium acetate buffer (pH 4) and 29.45 ± 4.84 μg/ml in MRS broth (pH 6.2). Drug-free IVRs and the released HCQ had no significant effects on bacterial growth or the viability of vaginal or ectocervical epithelial cells. Furthermore, there was no significant impact on the integrity of vaginal epithelial cell monolayers, in comparison with controls, as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the effects of HCQ-loaded IVRs on the growth of vaginal flora and the integrity of vaginal epithelial cell monolayers.

Yufei Chen,1 Yannick Leandre Traore,1 Amanda Li,1 Keith R Fowke,2,3 Emmanuel A Ho1 1Laboratory for Drug Delivery and Biomaterials, Faculty of Pharmacy, 2Department of Medical Microbiology and Infectious Diseases, 3Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada Abstract: Hydroxychloroquine (HCQ) has been shown to demonstrate anti-­inflammatory properties and direct anti-HIV activity. In this study, we describe for the first time the fabrication and in vitro evaluation of two types of intravaginal ring (IVR) devices (a surfaced-modified matrix IVR and a reservoir segmental IVR) for achieving sustained delivery (>14 days) of HCQ as a strategy for preventing male-to-female transmission of HIV. Both IVRs were fabricated by hot-melt injection molding. Surface-modified matrix IVRs with polyvinylpyrrolidone or poly(vinyl alcohol) coatings exhibited significantly reduced burst release on the first day (6.45% and 15.72% reduction, respectively). Reservoir IVR segments designed to release lower amounts of HCQ displayed near-zero-order release kinetics with an average release rate of 28.38 µg/mL per day for IVRs loaded with aqueous HCQ and 32.23 µg/mL per day for IVRs loaded with HCQ mixed with a rate-controlling excipient. Stability studies demonstrated that HCQ was stable in coated or noncoated IVRs for 30 days. The IVR segments had no significant effect on cell viability, pro-inflammatory cytokine production, or colony formation of vaginal and ectocervical epithelial cells. Both IVR systems may be suitable for the prevention of HIV transmission and other sexually transmitted infections. Keywords: intravaginal delivery, matrix system, reservoir system, polymeric drug carrier, drug release, microbicide, HIV/AIDS