Mesh and layered electrospun fiber architectures as vehicles for Lactobacillus acidophilus and Lactobacillus crispatus intended for vaginal delivery.

Bacterial vaginosis (BV) is a recurrent condition that affects millions of women worldwide. The use of probiotics is a promising alternative or an adjunct to traditional antibiotics for BV prevention and treatment. However, current administration regimens often require daily administration, thus contributing to low user adherence and recurrence. Here, electrospun fibers were designed to separately incorporate and sustain two lactic acid producing model organisms, Lactobacillus crispatus (L. crispatus) and Lactobacillus acidophilus (L. acidophilus). Fibers were made of polyethylene oxide and polylactic-co-glycolic acid in two different architectures, one with distinct layers and the other with co-spun components. Degradation of mesh and layered fibers was evaluated via mass loss and scanning electron microscopy. The results show that after 48 h and 6 days, cultures of mesh and layered fibers yielded as much as 108 and 109 CFU probiotic/mg fiber in total, respectively, with corresponding daily recovery on the order of 108 CFU/(mg·day). In addition, cultures of the fibers yielded lactic acid and caused a significant reduction in pH, indicating a high level of metabolic activity. The formulations did not affect vaginal keratinocyte viability or cell membrane integrity in vitro. Finally, mesh and layered probiotic fiber dosage forms demonstrated inhibition of Gardnerella, one of the most prevalent and abundant bacteria associated with BV, respectively resulting in 8- and 6.5-log decreases in Gardnerella viability in vitro after 24 h. This study provides initial proof of concept that mesh and layered electrospun fiber architectures developed as dissolving films may offer a viable alternative to daily probiotic administration.

Computational Modeling of Probiotic Recovery from 3D-Bioprinted Scaffolds for Localized Vaginal Application.

Lactobacilli, play a beneficial role in the female reproductive tract (FRT), regulating pH via lactic acid metabolism to help maintain a healthy environment. Bacterial vaginosis (BV) is characterized by a dysregulated flora in which anaerobes such as Gardnerella vaginalis (Gardnerella) create a less acidic environment. Current treatment focuses on antibiotic administration, including metronidazole, clindamycin, or tinidazole; however, lack of patient compliance as well as antibiotic resistance may contribute to 50% recurrence within a year. Recently, locally administered probiotics such as Lactobacillus crispatus (L. crispatus) have been evaluated as a prophylactic against recurrence. To mitigate the lack of patient compliance, sustained probiotic delivery has been proposed via 3D-bioprinted delivery vehicles. Successful delivery depends on a variety of vehicle fabrication parameters influencing timing and rate of probiotic recovery; detailed evaluation of these parameters would benefit from computational modeling complementary to experimental evaluation. This study implements a novel simulation platform to evaluate sustained delivery of probiotics from 3D-bioprinted scaffolds, taking into consideration bacterial lactic acid production and associated pH changes. The results show that the timing and rate of probiotic recovery can be realistically simulated based on fabrication parameters that affect scaffold degradation and probiotic survival. Longer term, the proposed approach could help personalize localized probiotic delivery to the FRT to advance women's health.

Rapid-dissolving electrospun nanofibers for intra-vaginal antibiotic or probiotic delivery.

The emergence of probiotics as an alternative and adjunct to antibiotic treatment for microbiological disturbances of the female genitourinary system requires innovative delivery platforms for vaginal applications. This study developed a new, rapid-dissolving form using electrospun polyethylene oxide (PEO) fibers for delivery of antibiotic metronidazole or probiotic Lactobacillus acidophilus, and performed evaluation in vitro and in vivo. Fibers did not generate overt pathophysiology or encourage Gardnerella growth in a mouse vaginal colonization model, inducing no alterations in vaginal mucosa at 24 hr post-administration. PEO-fibers incorporating metronidazole (100 µg MET/mg polymer) effectively prevented and treated Gardnerella infections (∼3- and 2.5-log reduction, respectively, 24 hr post treatment) when administered vaginally. Incorporation of live Lactobacillus acidophilus (107 CFU/mL) demonstrated viable probiotic delivery in vitro by PEO and polyvinyl alcohol (PVA) fibers to inhibit Gardnerella (108 CFU/mL) in bacterial co-cultures (9.9- and 7.0-log reduction, respectively, 24 hr post-inoculation), and in the presence of vaginal epithelial cells (6.9- and 8.0-log reduction, respectively, 16 hr post-inoculation). Administration of Lactobacillus acidophilus in PEO-fibers achieved vaginal colonization in mice similar to colonization observed with free Lactobacillus. acidophilus. These experiments provide proof-of-concept for rapid-dissolving electrospun fibers as a successful platform for intra-vaginal antibiotic or probiotic delivery.

Development and Characterization of Lactobacillus rhamnosus-Containing Bioprints for Application to Catheter-Associated Urinary Tract Infections.

Catheter-associated urinary tract infections (CAUTI) are a significant healthcare burden affecting millions of patients annually. CAUTI are characterized by infection of the bladder and pathogen colonization of the catheter surface, making them especially difficult to treat. Various catheter modifications have been employed to reduce pathogen colonization, including infusion of antibiotics and antimicrobial compounds, altering the surface architecture of the catheter, or coating it with nonpathogenic bacteria. Lactobacilli probiotics offer promise for a "bacterial interference" approach because they not only compete for adhesion to the catheter surface but also produce and secrete antimicrobial compounds effective against uropathogens. Three-dimensional (3D) bioprinting has enabled fabrication of well-defined, cell-laden architectures with tailored release of active agents, thereby offering a novel means for sustained probiotic delivery. Silicone has shown to be a promising biomaterial for catheter applications due to mechanical strength, biocompatibility, and its ability to mitigate encrustation on the catheter. Additionally, silicone, as a bioink, provides an optimum matrix for bioprinting lactobacilli. This study formulates and characterizes novel 3D-bioprinted Lactobacillus rhamnosus (L. rhamnosus)-containing silicone scaffolds for future urinary tract catheterization applications. Weight-to-weight (w/w) ratio of silicone/L. rhamnosus was bioprinted and cured with relative catheter dimensions in diameter. Scaffolds were analyzed in vitro for mechanical integrity, recovery of L. rhamnosus, antimicrobial production, and antibacterial effect against uropathogenic Escherichia coli, the leading cause of CAUTI. The results show that L. rhamnosus-containing scaffolds are capable of sustained recovery of live bacteria over 14 days, with sustained production of lactic acid and hydrogen peroxide. Through the use of 3D bioprinting, this study presents a potential alternative strategy to incorporate probiotics into urinary catheters, with the ultimate goal of preventing and treating CAUTI.

Release Kinetics of Metronidazole from 3D Printed Silicone Scaffolds for Sustained Application to the Female Reproductive Tract.

Sustained vaginal administration of antibiotics or probiotics has been proposed to improve treatment efficacy for bacterial vaginosis. 3D printing has shown promise for development of systems for local agent delivery. In contrast to oral ingestion, agent release kinetics can be fine-tuned by the 3D printing of specialized scaffold designs tailored for particular treatments while enhancing dosage effectiveness via localized sustained release. It has been challenging to establish scaffold properties as a function of fabrication parameters to obtain sustained release. In particular, the relationships between scaffold curing conditions, compressive strength, and drug release kinetics remain poorly understood. This study evaluates 3D printed scaffold formulation and feasibility to sustain the release of metronidazole, a commonly used antibiotic for BV. Cylindrical silicone scaffolds were printed and cured using three different conditions relevant to potential future incorporation of temperature-sensitive labile biologics. Compressive strength and drug release were monitored for 14d in simulated vaginal fluid to assess long-term effects of fabrication conditions on mechanical integrity and release kinetics. Scaffolds were mechanically evaluated to determine compressive and tensile strength, and elastic modulus. Release profiles were fitted to previous kinetic models to differentiate potential release mechanisms. The Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin models best described the release, indicating similarity to release from insoluble or polymeric matrices. This study shows the feasibility of 3D printed silicone scaffolds to provide sustained metronidazole release over 14d, with compressive strength and drug release kinetics tuned by the fabrication parameters.

Formulation and characterization of pressure-assisted microsyringe 3D-printed scaffolds for controlled intravaginal antibiotic release.

Bacterial vaginosis (BV) is a highly recurrent vaginal condition linked with many health complications. Topical antibiotic treatments for BV are challenged with drug solubility in vaginal fluid, lack of convenience and user adherence to daily treatment protocols, among other factors. 3D-printed scaffolds can provide sustained antibiotic delivery to the female reproductive tract (FRT). Silicone vehicles have been shown to provide structural stability, flexibility, and biocompatibility, with favorable drug release kinetics. This study formulates and characterizes novel metronidazole-containing 3D-printed silicone scaffolds for eventual application to the FRT. Scaffolds were evaluated for degradation, swelling, compression, and metronidazole release in simulated vaginal fluid (SVF). Scaffolds retained high structural integrity and sustained release. Minimal mass loss (<6%) and swelling (<2%) were observed after 14 days in SVF, relative to initial post-cure measurements. Scaffolds cured for 24 hr (50 °C) demonstrated elastic behavior under 20% compression and 4.0 N load. Scaffolds cured for 4 hr (50 °C), followed by 72 hr (4 °C), demonstrated the highest, sustained, metronidazole release (4.0 and 27.0 µg/mg) after 24 hr and 14 days, respectively. Based upon daily release profiles, it was observed that the 24 hr timepoint had the greatest metronidazole release of 4.08 µg/mg for scaffolds cured at 4 hr at 50 °C followed by 72 hr at 4 °C. For all curing conditions, release of metronidazole after 1 and 7 days showed > 4.0-log reduction in Gardnerella concentration. Negligible cytotoxicity was observed in treated keratinocytes comparable to untreated cells, This study shows that pressure-assisted microsyringe 3D-printed silicone scaffolds may provide a versatile vehicle for sustained metronidazole delivery to the FRT.

Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application.

Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. Post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.

Lactobacillus crispatus-loaded electrospun fibers yield viable and metabolically active bacteria that kill Gardnerella in vitro.

Bacterial vaginosis (BV) is a common condition that affects one-third of women worldwide. BV is characterized by low levels of healthy lactobacilli and an overgrowth of common anaerobes such as Gardnerella. Antibiotics for BV are administered orally or vaginally; however, approximately half of those treated will experience recurrence within 6 months. Lactobacillus crispatus present at high levels has been associated with positive health outcomes. To address the high recurrence rates following BV treatment, beneficial bacteria have been considered as an alternative or adjunct modality. This study aimed to establish proof-of-concept for a new long-acting delivery vehicle for L. crispatus. Here, it is shown that polyethylene oxide (PEO) fibers loaded with L. crispatus can be electrospun with poly(lactic-co-glycolic acid) (PLGA) fibers (ratio 1:1), and that this construct later releases L. crispatus as metabolically viable bacteria capable of lactic acid production and anti-Gardnerella activity. Probiotic-containing fibers were serially cultured in MRS (deMan, Rogosa, Sharpe) broth with daily media replacement and found to yield viable L. crispatus for at least 7 days. Lactic acid levels and corresponding pH values generally corresponded with levels of L. crispatus cultured from the fibers and strongly support the conclusion that fibers yield viable L. crispatus that is metabolically active. Cultures of L. crispatus-loaded fibers limited the growth of Gardnerella in a dilution-dependent manner during in vitro assays in the presence of cultured vaginal epithelial cells, demonstrating bactericidal potential. Exposure of VK2/E6E7 cells to L. crispatus-loaded fibers resulted in minimal loss of viability relative to untreated cells. Altogether, these data provide proof-of-concept for electrospun fibers as a candidate delivery vehicle for application of vaginal probiotics in a long-acting form.

Multicellular Ovarian Cancer Model for Evaluation of Nanovector Delivery in Ascites and Metastatic Environments.

A novel multicellular model composed of epithelial ovarian cancer and fibroblast cells was developed as an in vitro platform to evaluate nanovector delivery and ultimately aid the development of targeted therapies. We hypothesized that the inclusion of peptide-based scaffold (PuraMatrix) in the spheroid matrix, to represent in vivo tumor microenvironment alterations along with metastatic site conditions, would enhance spheroid cell growth and migration and alter nanovector transport. The model was evaluated by comparing the growth and migration of ovarian cancer cells exposed to stromal cell activation and tissue hypoxia. Fibroblast activation was achieved via the TGF-ß1 mediated pathway and tissue hypoxia via 3D spheroids incubated in hypoxia. Surface-modified nanovector transport was assessed via fluorescence and confocal microscopy. Consistent with previous in vivo observations in ascites and at distal metastases, spheroids exposed to activated stromal microenvironment were denser, more contractile and with more migratory cells than nonactivated counterparts. The hypoxic conditions resulted in negative radial spheroid growth over 5 d compared to a radial increase in normoxia. Nanovector penetration attenuated in PuraMatrix regardless of surface modification due to a denser environment. This platform may serve to evaluate nanovector transport based on ovarian ascites and metastatic environments, and longer term, it provide a means to evaluate nanotherapeutic efficacy.

Perspectives on Existing and Novel Alternative Intravaginal Probiotic Delivery Methods in the Context of Bacterial Vaginosis Infection.

Bacterial vaginosis (BV) is one of the most common vaginal infections that affects hundreds of millions of women of reproductive age, worldwide. Traditional treatment strategies, such as oral and topical antibiotics, have shown efficacy against BV, but frequent recurrence of infection and the development of antibiotic-resistant bacteria remain as significant challenges. Alternatively, recent progress in understanding immune, microbiological, and metabolic interactions in the vaginal microbiota has prompted the consideration of administering probiotic organisms to restore and maintain vaginal health within the context of BV prevention and treatment. Given this, the objective of this review is to discuss existing and potential alternative approaches to deliver, and to potentially sustain the delivery of probiotics, to prevent and/or treat BV infections. First, a brief overview is provided regarding the probiotic species and combinatorial probiotic strategies that have shown promise in the treatment of BV and in restoring female reproductive health. Additionally, the advantages and challenges associated with current oral and intravaginal probiotic delivery platforms are discussed. Lastly, we present emerging and promising alternative dosage forms, such as electrospun fibers and 3D bioprinted scaffolds, that may be adapted as new strategies to intravaginally deliver probiotic organisms. Graphical abstract.

In vitro Study on Synergistic Interactions Between Free and Encapsulated Q-Griffithsin and Antiretrovirals Against HIV-1 Infection.

INTRODUCTION: Human immunodeficiency virus (HIV) remains a persistent global challenge, impacting 38 million people worldwide. Antiretrovirals (ARVs) including tenofovir (TFV), raltegravir (RAL), and dapivirine (DAP) have been developed to prevent and treat HIV-1 via different mechanisms of action. In parallel, a promising biological candidate, griffithsin (GRFT), has demonstrated outstanding preclinical safety and potency against HIV-1. While ARV co-administration has been shown to enhance virus inhibition, synergistic interactions between ARVs and the oxidation-resistant variant of GRFT (Q-GRFT) have not yet been explored. Here, we co-administered Q-GRFT with TFV, RAL, and DAP, in free and encapsulated forms, to identify unique protein-drug synergies. METHODS: Nanoparticles (NPs) were synthesized using a single or double-emulsion technique and release from each formulation was assessed in simulated vaginal fluid. Next, each ARV, in free and encapsulated forms, was co-administered with Q-GRFT or Q-GRFT NPs to evaluate the impact of co-administration in HIV-1 pseudovirus assays, and the combination indices were calculated to identify synergistic interactions. Using the most synergistic formulations, we investigated the effect of agent incorporation in NP-fiber composites on release properties. Finally, NP safety was assessed in vitro using MTT assay. RESULTS: All active agents were encapsulated in NPs with desirable encapsulation efficiency (15-100%), providing ~20% release over 2 weeks. The co-administration of free Q-GRFT with each free ARV resulted in strong synergistic interactions, relative to each agent alone. Similarly, Q-GRFT NP and ARV NP co-administration resulted in synergy across all formulations, with the most potent interactions between encapsulated Q-GRFT and DAP. Furthermore, the incorporation of Q-GRFT and DAP in NP-fiber composites resulted in burst release of DAP and Q-GRFT with a second phase of Q-GRFT release. Finally, all NP formulations exhibited safety in vitro. CONCLUSIONS: This work suggests that Q-GRFT and ARV co-administration in free or encapsulated forms may improve efficacy in achieving prophylaxis.

Assessment of CafA Targeted BAR-Encapsulated Nanoparticles against Oral Biofilms.

Porphyromonas gingivalis adherence to Streptococcus gordonii is a crucial initial event that facilitates the colonization of P. gingivalis, a key pathogen in periodontal disease. As such, blocking these early interactions may present a potential avenue to limit P. gingivalis colonization. Nanoparticles encapsulating a synthetic peptide BAR (BAR-encapsulated NPs) inhibit P. gingivalis/S. gordonii biofilm formation 1.8-fold more potently relative to free BAR. However, BAR-encapsulated NPs, like many orally delivered formulations, may benefit from a strategy that improves their retention in an open flow environment. Here, we sought to enhance the efficacy of BAR-encapsulated NPs by modifying their surfaces with coaggregation factor A (CafA), a fimbrial protein expressed by the early colonizer, Actinomyces oris. We demonstrate that the targeting moiety, CafA, enhances NP binding and exhibits specificity of adherence to S. gordonii, relative to other oral bacterial species. Furthermore, CafA-modified NPs release inhibitory concentrations of BAR for 12 h, a time frame relevant to oral dosage form delivery. Lastly, CafA-modified NPs potently inhibit P. gingivalis/S. gordonii biofilm formation for up to 12 h and are non-toxic at therapeutically-relevant concentrations. These results suggest that CafA-modified NPs represent a novel and efficacious delivery vehicle for localized, targeted delivery of BAR to P. gingivalis preferred niches.

Rapid-Release Griffithsin Fibers for Dual Prevention of HSV-2 and HIV-1 Infections.

The biologic griffithsin (GRFT) has recently emerged as a candidate to safely prevent sexually transmitted infections (STIs), including human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus 2 (HSV-2). However, to date, there are few delivery platforms that are available to effectively deliver biologics to the female reproductive tract (FRT). The goal of this work was to evaluate rapid-release polyethylene oxide (PEO), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) fibers that incorporate GRFT in in vitro (HIV-1 and HSV-2) and in vivo (HSV-2) infection models. GRFT loading was determined via enzyme-linked immunosorbent assay (ELISA), and the bioactivity of GRFT fibers was assessed using in vitro HIV-1 pseudovirus and HSV-2 plaque assays. Afterwards, the efficacy of GRFT fibers was assessed in a murine model of lethal HSV-2 infection. Finally, murine reproductive tracts and vaginal lavage samples were evaluated for histology and cytokine expression, 24 and 72 h after fiber administration, to determine safety. All rapid-release formulations achieved high levels of GRFT incorporation and were completely efficacious against in vitro HIV-1 and HSV-2 infections. Importantly, all rapid-release GRFT fibers provided potent protection in a murine model of HSV-2 infection. Moreover, histology and cytokine levels, evaluated from collected murine reproductive tissues and vaginal lavage samples treated with blank fibers, showed no increased cytokine production or histological aberrations, demonstrating the preliminary safety of rapid-release GRFT fibers in vaginal tissue.

Sustained-release Griffithsin nanoparticle-fiber composites against HIV-1 and HSV-2 infections.

Human immunodeficiency virus (HIV-1) and herpes simplex virus 2 (HSV-2) affect hundreds of millions of people worldwide. The antiviral lectin, Griffithsin (GRFT), has been shown to be both safe and efficacious against HSV-2 and HIV-1 infections in vivo. The goal of this work was to develop a multilayered nanoparticle (NP)-electrospun fiber (EF) composite to provide sustained-release of GRFT, and to examine its safety and efficacy in a murine model of lethal HSV-2 infection. Composites were fabricated from polycaprolactone (PCL) fibers surrounding polyethylene oxide (PEO) fibers that incorporated methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) GRFT NPs. GRFT loading and release were determined via ELISA, showing that NP-EF composites achieved high GRFT loading, and provided sustained-release of GRFT for up to 90 d. The in vitro efficacy of GRFT NP-EFs was assessed using HIV-1 pseudovirus assays, demonstrating complete in vitro protection against HIV-1 infection. Additionally, sustained-release NP-EFs, administered 24 h prior to infection, prevented against a lethal dose of HSV-2 infection in a murine model. In parallel, histology and cytokine expression from murine reproductive tracts and vaginal lavages collected 24 and 72 h post-administration were similar to untreated mice, suggesting that NP-EF composites may be a promising and safe sustained-delivery platform to prevent HSV-2 infection. Future work will evaluate the ability to provide prolonged protection against multiple virus challenges, and different administration times with respect to infection.

Surface-Modified Melphalan Nanoparticles for Intravitreal Chemotherapy of Retinoblastoma.

Purpose: The goal of this work was to design and assess the ability of unmodified and surface-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to enhance cell association, provide efficacy in retinoblastoma cells, and overcome current administration challenges, including hydrolysis and precipitation, of intravitreal administration. Methods: A single emulsion method was used to encapsulate Coumarin 6, to enable NP visualization via fluorescence microscopy. Melphalan NPs were synthesized using an adapted double-emulsion method to reduce melphalan loss during fabrication. Melphalan loading and release were quantified against a free melphalan standard. The cellular association and internalization of unmodified and surface-modified NPs were determined using flow cytometry, and the efficacy of melphalan NPs was quantified in retinoblastoma cells. Results: The highest cell association was observed with TET1 and MPG-NPs after 24 hours administration; however, a significant fraction of NPs were associated with the cell surface, instead of undergoing internalization. MPG-NPs fabricated with the low saturation process were most efficacious, while all surface-modified NPs improved efficacy relative to unmodified NPs when formulated using the highly saturated process. Similar effects were observed as a function of NP dose, with TET1 and MPG-NPs particularly efficacious. Conclusions: Surface-modified NPs achieved enhanced association and efficacy in retinoblastoma cells relative to unmodified NPs, with MPG and surface-modified NPs exhibiting the strongest efficacy relative to other NP groups. In future work we seek to assess the ability of these NPs to improve transport in the vitreous, where we expect a more dramatic impact on efficacy as a function of surface modification.

Relating Advanced Electrospun Fiber Architectures to the Temporal Release of Active Agents to Meet the Needs of Next-Generation Intravaginal Delivery Applications.

Electrospun fibers have emerged as a relatively new delivery platform to improve active agent retention and delivery for intravaginal applications. While uniaxial fibers have been explored in a variety of applications including intravaginal delivery, the consideration of more advanced fiber architectures may offer new options to improve delivery to the female reproductive tract. In this review, we summarize the advancements of electrospun coaxial, multilayered, and nanoparticle-fiber architectures utilized in other applications and discuss how different material combinations within these architectures provide varied durations of release, here categorized as either transient (within 24 h), short-term (24 h to one week), or sustained (beyond one week). We seek to systematically relate material type and fiber architecture to active agent release kinetics. Last, we explore how lessons derived from these architectures may be applied to address the needs of future intravaginal delivery platforms for a given prophylactic or therapeutic application. The overall goal of this review is to provide a summary of different fiber architectures that have been useful for active agent delivery and to provide guidelines for the development of new formulations that exhibit release kinetics relevant to the time frames and the diversity of active agents needed in next-generation multipurpose applications.

Efficacy of Surface-Modified PLGA Nanoparticles as a Function of Cervical Cancer Type.

PURPOSE: Hypovascularization of cervical tumors, coupled with intrinsic and acquired drug resistance, has contributed to marginal therapeutic outcomes by hindering chemotherapeutic transport and efficacy. Recently, the heterogeneous penetration and distribution of cell penetrating peptide (CPP, here MPG) and polyethylene glycol (PEG) modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were evaluated as a function of tumor type and morphology in cervical cancer spheroids modeling hypovascularized tumor nodules. Building upon this work, this study investigates the efficacy imparted by surface-modified Doxorubicin-loaded NPs transported into hypovascularized tissue. METHODS: NP efficacy was measured in HeLa, CaSki, and SiHa cells. NP internalization and association, and associated cell viability, were determined in monolayer and spheroid models. RESULTS: MPG and PEG-NP co-treatment was most efficacious in HeLa cells, while PEG NPs were most efficacious in CaSki cells. NP surface-modifications were unable to improve efficacy, relative to unmodified NPs, in SiHa cells. CONCLUSIONS: The results highlight the dependence of efficacy on tumor type and the associated microenvironment. The results further relate previous NP transport studies to efficacy, as a function of surface-modification and cell type. Longer-term, this information may help guide the design of NP-mediated strategies to maximize efficacy based on patient-specific cervical tumor origin and characteristics.

Functional assessment of peptide-modified PLGA nanoparticles against oral biofilms in a murine model of periodontitis.

The interaction of the periodontal pathogen Porphyromonas gingivalis (Pg) with commensal streptococci promotes Pg colonization of the oral cavity. Previously, we demonstrated that a peptide (BAR) derived from Streptococcus gordonii (Sg) potently inhibited adherence of Pg to streptococci and reduced Pg virulence in a mouse model of periodontitis. Thus, BAR may represent a novel therapeutic to control periodontitis by preventing Pg colonization of the oral cavity. However, while BAR inhibited the initial formation of Pg/Sg biofilms, much higher concentrations of peptide were required to disrupt an established Pg/Sg biofilm. To improve the activity of the peptide, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were surface-modified with BAR and shown to more potently disrupt Pg/Sg biofilms relative to an equimolar amount of free peptide. The goal of this work was to determine the in vivo efficacy of BAR-modified NPs (BNPs) and to assess the toxicity of BNPs against human gingival epithelial cells. In vivo efficacy of BNPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Infection of mice with Pg and Sg resulted in a significant increase in alveolar bone loss and gingival IL-17 expression over sham-infected animals. Treatment of Pg/Sg infected mice with BNPs reduced bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The cytotoxicity of the maximum concentration of BNPs and free BAR used in in vitro and in vivo studies (1.3 and 3.4 µM), was evaluated in telomerase immortalized gingival keratinocytes (TIGKs) by measuring cell viability, cell lysis and apoptosis. BNPs were also tested for hemolytic activity against sheep erythrocytes. TIGKs treated with BNPs or free BAR demonstrated >90% viability and no significant lysis or apoptosis relative to untreated cells. In addition, neither BNPs nor free BAR exhibited hemolytic activity. In summary, BNPs were non-toxic within the evaluated concentration range of 1.3-3.4 µM and provided more efficacious protection against Pg-induced inflammation in vivo, highlighting the potential of BNPs as a biocompatible platform for translatable oral biofilm applications.

pH-responsive delivery of Griffithsin from electrospun fibers.

Human immunodeficiency virus (HIV-1) affects over 36 million people globally. Current prevention strategies utilize antiretrovirals that have demonstrated protection, but result in antiviral resistance, adverse toxicity, and require frequent administration. A novel biologic, griffithsin (GRFT), has demonstrated outstanding safety and efficacy against laboratory and primary HIV isolates and against intravaginal murine herpes simplex virus 2 (HSV-2) challenge, making it a promising microbicide candidate. However, transient activity and instability remain concerns surrounding biologic delivery, particularly in the harsh environment of the female reproductive tract (FRT). Recently, electrospun fibers (EFs) have demonstrated promise for intravaginal delivery, with the potential to conserve active agent until release is needed. The goal of this study was to fabricate and characterize pH-responsive fibers comprised of poly(lactic-co-glycolic acid) (PLGA) or methoxypolyethylene glycol-b-PLGA (mPEG-PLGA) with varying ratios of poly(n-butyl acrylate-co-acrylic acid) (PBA-co-PAA), to selectively release GRFT under pH-conditions that mimic semen introduction. Fibers comprised of mPEG-PLGA:PBA-co-PAA (90:10 w/w) demonstrated high GRFT loading that was maintained within simulated vaginal fluid (SVF), and pH-dependent release upon exposure to buffered and SVF:simulated semen solutions. Moreover, GRFT fibers demonstrated potent in vitro efficacy against HIV-1 and safety in vaginal epithelial cells, suggesting their future potential for efficacious biologic delivery to the FRT.

Glycoprotein-130 Expression Is Associated with Aggressive Bladder Cancer and Is a Potential Therapeutic Target.

Predicting bladder cancer progression is important in selecting the optimal treatment for bladder cancer. Because current diagnostic factors regarding progression are lacking, new factors are needed to further stratify the curative potential of bladder cancer. Glycoprotein-130 (GP130), a transmembrane protein, is central to a number of signal transduction pathways involved in tumor aggressiveness, making it an attractive target. We hypothesize that if GP130 is found in an aggressive population of bladder tumors, then blocking GP130 expression may inhibit bladder cancer growth. Herein, we quantitatively show, using 11 patient samples and four bladder cancer cell lines, that GP130 is expressed in the aggressive human bladder tumors and in high-grade bladder cancer cell lines. Moreover, GP130 is significantly correlated with tumor grade, node category, tumor category, and patient outcome. We demonstrated a tumor-specific GP130 effect by blocking GP130 expression in bladder tumor cells, which resulted in decreased cell viability and reduced cell migration. Furthermore, we reduced tumor volume by approximately 70% compared with controls by downregulating GP130 expression using chitosan-functionalized nanoparticles encapsulating GP130 siRNA in an in vivo bladder cancer xenograft mouse model. Our results indicate that GP130 expression is linked to the aggressiveness of bladder tumors, and blocking GP130 has therapeutic potential in controlling tumor growth.