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Farnaz Minooei,1 Joel R Fried,1 Joshua L Fuqua,2– 4 Kenneth E Palmer,3– 5 Jill M Steinbach-Rankins2– 5 1Department of Chemical Engineering, University of Louisville Speed School of Engineering, Louisville, KY, USA; 2Department of Bioengineering, University of Louisville Speed School of Engineering, Louisville, KY, USA; 3Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA; 4Center for Predictive Medicine, University of Louisville, Louisville, KY, USA; 5Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY, USACorrespondence: Jill M Steinbach-RankinsDepartment of Bioengineering, University of Louisville Speed School of Engineering, 505 S. Hancock St., Room 623, Louisville, KY, 40202, USATel +1 502-852-5486Email jmstei01@louisville.eduIntroduction: 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.Keywords: griffithsin, nanoparticles, electrospun fibers, antiretrovirals, synergy, HIV-1 prevention, microbicide

Lee B Sims,1 Hermann B Frieboes,1–3 Jill M Steinbach-Rankins1,3–5 1Department of Bioengineering, University of Louisville, Louisville, KY, USA; 2James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA; 3Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA; 4Department of Microbiology and Immunology, University of Louisville, Louisville, KY, USA; 5Center for Predictive Medicine, University of Louisville, Louisville, KY, USA Abstract: 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. Keywords: polymer nanoparticles, 3D cell culture, cervicovaginal mucus, CVM, transport, female reproductive tract, FRT, mathematical modeling, intravaginal delivery

Paridhi Kalia,1 Ankita Jain,1 Ranjith Radha Krishnan,1 Donald R Demuth,1,2 Jill M Steinbach-Rankins2–5 1Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, 2Department of Microbiology and Immunology, University of Louisville School of Medicine, 3Department of Bioengineering, University of Louisville Speed School of Engineering, 4Department of Pharmacology and Toxicology, University of Louisville School of Medicine, 5Center for Predictive Medicine, University of Louisville, Louisville, KY, USA Purpose: The interaction of Porphyromonas gingivalis with commensal streptococci promotes P. gingivalis colonization of the oral cavity. We previously showed that a synthetic peptide (BAR) derived from Streptococcus gordonii potently inhibited the formation of P. gingivalis/S. gordonii biofilms (IC50 =1.3 µM) and reduced P. gingivalis virulence in a mouse model of periodontitis. Thus, BAR represents a novel therapeutic to control periodontitis by limiting P. gingivalis colonization of the oral cavity. Here, we sought to develop drug-delivery vehicles for potential use in the oral cavity that comprise BAR-modified poly(lactic-co-glycolic)acid (PLGA) nanoparticles (NPs). Methods: PLGA-NPs were initially modified with palmitylated avidin and subsequently conjugated with biotinylated BAR. The extent of BAR modification was quantified using a fluorescent-labeled peptide. Inhibition of P. gingivalis adherence to S. gordonii by BAR-modified NPs was compared with free peptide using a two-species biofilm model. Results: BAR-modified NPs exhibited an average size of 99±29 nm and a more positive surface charge than unmodified NPs (zeta potentials of -7 mV and -25 mV, respectively). Binding saturation occurred when 37 nmol BAR/mg of avidin-NPs was used, which resulted in a payload of 7.42 nmol BAR/mg NPs. BAR-modified NPs bound to P. gingivalis in a dose-dependent manner and more potently inhibited P. gingivalis/S. gordonii adherence and biofilm formation relative to an equimolar amount of free peptide (IC50 of 0.2 µM versus 1.3 µM). BAR-modified NPs also disrupted the preformed P. gingivalis/S. gordonii biofilms more effectively than free peptide. Finally, we demonstrate that BAR-modified NPs promoted multivalent association with P. gingivalis, providing an explanation for the increased effectiveness of NPs. Conclusion: These results indicate that BAR-modified NPs deliver a higher local dose of peptide and may represent a more effective therapeutic approach to limit P. gingivalis colonization of the oral cavity compared to treatment with formulations of free peptide. Keywords: nanoparticle, peptide delivery, multivalent, drug delivery, Porphyromonas gingivalis, periodontal disease

To date, the commonly used methods for diffusion coefficient measurements have some hurdles that prevent them from being widely applied in pharmaceutical laboratories. This study aimed to modify a method developed by di Cagno et al. based on the use of a UV-Vis spectrometer and apply the method to investigate the effect of dissolution media on the diffusivity of small molecules and proteins. A total of five small molecules and two proteins in different aqueous media and polymer solutions were investigated in this study. By attaching a 3D-printed cover with an open slit to a standard UV-Vis cuvette, the incident UV light could only pass through the open slit to measure the local drug concentration. During the diffusion experiment, drug molecules diffused from the cuvette bottom to the slit. According to the concentration measured as a function of time, diffusion coefficient was calculated based on Fick's law of diffusion using the analytical and numerical approaches. As a result, diffusion coefficients could be accurately measured with high reproducibility. The results also suggested that different media could affect the diffusion coefficients of small molecules by < 10% and proteins by < 15%. Since the UV-Vis spectrometer is a routine instrument, this method can potentially be employed by many pharmaceutical laboratories for diffusion coefficient measurements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The acidic byproducts of bacteria in plaque around orthodontic brackets contribute to white spot lesion (WSL) formation. Nitric oxide (NO) has antibacterial properties, hindering biofilm formation and inhibiting the growth of oral microbes. Materials that mimic NO release could prevent oral bacteria-related pathologies. This study aims to integrate S-nitroso-acetylpenicillamine (SNAP), a promising NO donor, into orthodontic elastomeric ligatures, apply an additional polymer coating, and evaluate the NO-release kinetics and antimicrobial activity against Streptococus mutans . SNAP was added to clear elastomeric chains (8 loops, 23 mm long) at three concentrations (50, 75, 100 mg/mL, and a control). Chains were then coated, via electrospinning, with additional polymer (Elastollan ® ) to aid in extending the NO release. NO flux was measured daily for 30 days. Samples with 75 mg/mL SNAP + Elastollan ® were tested against S. mutans for inhibition of biofilm formation on and around the chain. SNAP was successfully integrated into ligatures at each concentration. Only the 75 mg/mL SNAP chains maintained their elasticity. After polymer coating, samples exhibited a significant burst of NO on the first day, exceeding the machine's reading capacity, which gradually decreased over 29 days. Ligatures also inhibited S. mutans growth and biofilm formation. Future research will assess their mechanical properties and cytotoxicity. This study presents a novel strategy to address white spot lesion (WSL) formation and bacterial-related pathologies by utilizing nitric oxide-releasing materials. Manufactured chains with antimicrobial properties provide a promising solution for orthodontic challenges, showing significant potential for academic-industrial collaboration and commercial viability.

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 10 8 and 10 9  CFU probiotic/mg fiber in total, respectively, with corresponding daily recovery on the order of 10 8  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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 (10 7 CFU/mL) demonstrated viable probiotic delivery in vitro by PEO and polyvinyl alcohol (PVA) fibers to inhibit Gardnerella (10 8 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of Interests Statement The authors declare no known conflicts of interest.

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.

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., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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-β1 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.

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.

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.

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., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Active agents targeting key bacterial interactions that initiate biofilm formation in the oral cavity, may alter periodontitis progression; however, to date, specifically-targeted prophylactic and treatment strategies have been limited. Previously we developed a peptide, BAR (SspB Adherence Region), that inhibits oral P. gingivalis/S. gordonii biofilm formation in vitro and in vivo , and BAR nanoparticles that increase BAR effectiveness via multivalency and prolonged delivery. However, limited BAR loading and nanoparticle retention in the oral cavity can result in inadequate release and efficaciousness. Given this, an effective delivery platform that can release concentrations of BAR suitable for twice-daily applications, may offer an alternative that enhances loading, ease of administration, and retention in the oral cavity. With this in mind, the study objectives were to develop and characterize a rapid-release platform, composed of polymeric electrospun fibers (EFs) that encapsulate BAR, and to evaluate fiber safety and functionality against P. gingivalis / S. gordonii biofilms in vitro . Poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic acid) (PLLA), and polycaprolactone (PCL) were electrospun alone or blended with polyethylene oxide (PEO), to provide high BAR loading and rapid-release. The most promising formulation, 10:90 PLGA:PEO EFs, provided 95% BAR release after 4 h, dose-dependent inhibition of biofilm formation (IC50 = 1.3 μM), disruption of established dual-species biofilms (IC50 = 2 μM), and maintained high cell viability. These results suggest that BAR-incorporated EFs may provide a safe and specifically-targeted rapid-release platform to inhibit and disrupt dual-species biofilms, that we envision may be applied twice-daily to exert prophylactic effect in the oral cavity., (Copyright © 2020 Mahmoud, Sapare, Curry, Demuth and Steinbach-Rankins.)

The secreted mucus layer in the vaginal epithelium presents a formidable barrier to the transport of active agents for the prevention and treatment of female reproductive tract (FRT) infections. Nanoparticle-mediated drug delivery has been proposed to help facilitate the transport and release of active agents through the cervicovaginal mucus (CVM) and underlying mucosa. However, both nanoparticles (NPs) and free active agents face a variety of challenges, often requiring the administration of high localized doses to circumvent leakage and poor penetration to targeted intravaginal tissue compartments. To address these challenges, "stealth" NP modifications have been investigated, due to their favorable mucus-penetrating properties, resulting in improved intravaginal active agent retention and transport. A number of other NP characteristics including size, surface modification type, ligand density, and co-modification, as well as the complexity of the FRT tissue are involved in obtaining adequate tissue penetration and, if needed, cell internalization. Studies that systematically investigate variations of these characteristics have yet to be conducted, with the goal to obtain a better understanding of what properties most impact prophylactic and therapeutic benefit. To complement the progress made with experimental evaluation of active agent transport in in vitro and in vivo, mathematical modeling has recently been applied to analyze the transport performance of agents and delivery vehicles in the FRT. Here, we build upon this work to simulate NP transport through mucus gel, epithelial, and stromal compartments, with the goal to provide a platform that can systematically evaluate transport based on NP and tissue characteristics. Model parameters such as PEG density and NP release (decay) rate from mucus gel into the epithelium, are set from previous in vitro and in vivo experimental work that assessed the transport of poly(lactic-co-glycolic acid (PLGA) NPs. The modeling results show that while unmodified and 2% PEG-modified NPs were retained in mucus for ∼1-4 h, dependent upon decay constant values, and traverse to the epithelium, no NP penetration was observed in the stroma. In contrast, NPs modified with 3% PEG, exhibited prolonged retention in each compartment, remaining for ∼4-6 h. Moreover, a significant concentration of NPs is observed in the stroma, indicating a transition in transport behavior. For NPs modified with 5, 8, or 25% PEG, steady retention profiles were noted, which gradually decline over 24 h. To supplement this modeling study and to develop a more representative experimental system that may be useful in future work, we report on the feasibility of constructing single and multicellular layered (MCL) culture systems to represent the epithelial and stromal tissue of the FRT. We anticipate that a combined mathematical/experimental approach may longer term enable prediction and customization of patient tissue-specific approaches to attain effective NP-mediated drug delivery and release for the treatment of infectious disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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.

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (©2018 American Association for Cancer Research.)

Background: Porphyromonas gingivalis adherence to oral streptococci is a key point in the pathogenesis of periodontal diseases (Honda in Cell Host Microbe 10:423-425, 2011). Previous work in our groups has shown that a region of the streptococcal antigen denoted BAR (SspB Adherence Region) inhibits P. gingivalis/S. gordonii interaction and biofilm formation both in vitro and in a mouse model of periodontitis (Daep et al. in Infect Immun 74:5756-5762, 2006; Daep et al. in Infect immun 76:3273-3280, 2008; Daep et al. in Infect Immun 79:67-74, 2011). However, high localized concentration and prolonged exposure are needed for BAR to be an effective therapeutic in the oral cavity., Methods: To address these challenges, we fabricated poly(lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol PLGA (mPEG-PLGA) nanoparticles (NPs) that encapsulate BAR peptide, and assessed the potency of BAR-encapsulated NPs to inhibit and disrupt in vitro two-species biofilms. In addition, the kinetics of BAR-encapsulated NPs were compared after different durations of exposure in a two-species biofilm model, against previously evaluated BAR-modified NPs and free BAR., Results: BAR-encapsulated PLGA and mPEG-PLGA NPs potently inhibited biofilm formation (IC50 = 0.7 μM) and also disrupted established biofilms (IC50 = 1.3 μM) in a dose-dependent manner. In addition, BAR released during the first 2 h of administration potently inhibits biofilm formation, while a longer duration of 3 h is required to disrupt pre-existing biofilms., Conclusions: These results suggest that BAR-encapsulated NPs provide a potent platform to inhibit (prevent) and disrupt (treat) P. gingivalis/S. gordonii biofilms, relative to free BAR.

The need for more versatile technologies to deliver antiviral agents to the female reproductive tract (FRT) has spurred the development of on-demand and sustained-release platforms. Electrospun fibers (EFs), in particular, have recently been applied to FRT delivery, resulting in an alternative dosage form with the potential to provide protection and therapeutic effect against a variety of infection types. However, a multitude of fabrication parameters, as well as the resulting complexities of solvent-drug, drug-polymer, and solvent-polymer interactions, are known to significantly impact the loading and release of incorporated agents. Numerous processing parameters, in addition to their combined interactions, can hinder the iterative development of fiber formulations to achieve optimal release for particular durations, doses, and polymer-drug types. The experimental effort to design and develop EFs could benefit from mathematical analysis and computational simulation that predictively evaluate combinations of parameters to meet product design needs. Here, existing modeling efforts are leveraged to develop a simulation platform that correlates and predicts the delivery of relevant small molecule antivirals from EFs that have been recently applied to target sexually transmitted infections (STIs). A pair of mathematical models is coupled to simulate the release of two structurally similar small molecule antiretroviral reverse transcriptase inhibitors, Tenofovir (TFV) and Tenofovir disoproxil fumarate (TDF), from poly(lactic- co-glycolic acid) (PLGA) EFs, and to evaluate how changes in the system parameters affect the distribution of encapsulated agent in a three-compartment model of the vaginal epithelium. The results indicate that factors such as fiber diameter, mesh thickness, antiviral diffusivity, and fiber geometry can be simulated to create an accurate model that distinguishes the different release patterns of TFV and TDF from EFs, and that enables detailed evaluation of the associated pharmacokinetics. This simulation platform offers a basis with which to further study EF parameters and their effect on antiviral release and pharmacokinetics in the FRT.

Nanoparticle and other drug delivery platforms have demonstrated promising potential for the delivery of therapeutics or imaging agents in a specific and targeted manner. While a variety of drug delivery platforms have been applied to medicine, in vitro and in silico optimization and validation of these targeting constructs needs to be conducted to maximize in vivo delivery and efficacy. Here, we describe the mathematical and experimental models to predict and validate the transport of a peptide targeting construct through a mock tissue environment to specifically target tumor cells, relative to non-tumor cells. We provide methods to visualize and analyze fluorescence microscopy images, and also describe the methods for creating a finite element model (FEM) that validates important parameters of this experimental system. By comparing and contrasting mathematical modeling results with experimental results, important information can be imparted to the design and functionality of the targeting construct. This information will help to optimize construct design for future therapeutic delivery applications.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

Sexually transmitted infections affect hundreds of millions of people worldwide. Both human immunodeficiency virus (HIV-1 and -2) and herpes simplex virus-2 (HSV-2) remain incurable, urging the development of new prevention strategies. While current prophylactic technologies are dependent on strict user adherence to achieve efficacy, there is a dearth of delivery vehicles that provide discreet and convenient administration, combined with prolonged-delivery of active agents. To address these needs, we created electrospun fibers (EFs) comprised of FDA-approved polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(DL-lactide-co-ε-caprolactone) (PLCL), to provide sustained-release and in vitro protection against HIV-1 and HSV-2. PLGA and PLCL EFs, incorporating the antiretroviral, tenofovir disoproxil fumarate (TDF), exhibited sustained-release for up to 4 weeks, and provided complete in vitro protection against HSV-2 and HIV-1 for 24h and 1 wk, respectively, based on the doses tested. In vitro cell culture and EpiVaginal tissue tests confirmed the safety of fibers in vaginal and cervical cells, highlighting the potential of PLGA and PLCL EFs as multipurpose next-generation drug delivery vehicles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Advanced stage cancer treatments are often invasive and painful-typically comprised of surgery, chemotherapy, and/or radiation treatment. Low transport efficiency during systemic chemotherapy may require high chemotherapeutic doses to effectively target cancerous tissue, resulting in systemic toxicity. Nanotherapeutic platforms have been proposed as an alternative to more safely and effectively deliver therapeutic agents directly to tumor sites. However, cellular internalization and tumor penetration are often diametrically opposed, with limited access to tumor regions distal from vasculature, due to irregular tissue morphologies. To address these transport challenges, nanoparticles (NPs) are often surface-modified with ligands to enhance transport and longevity after localized or systemic administration. Here, we evaluate stealth polyethylene-glycol (PEG), cell-penetrating (MPG), and CPP-stealth (MPG/PEG) poly(lactic-co-glycolic-acid) (PLGA) NP co-treatment strategies in 3D cell culture representing hypo-vascularized tissue., Results: Smaller, more regularly-shaped avascular tissue was generated using the hanging drop (HD) method, while more irregularly-shaped masses were formed with the liquid overlay (LO) technique. To compare NP distribution differences within the same type of tissue as a function of different cancer types, we selected HeLa, cervical epithelial adenocarcinoma cells; CaSki, cervical epidermoid carcinoma cells; and SiHa, grade II cervical squamous cell carcinoma cells. In HD tumors, enhanced distribution relative to unmodified NPs was measured for MPG and PEG NPs in HeLa, and for all modified NPs in SiHa spheroids. In LO tumors, the greatest distribution was observed for MPG and MPG/PEG NPs in HeLa, and for PEG and MPG/PEG NPs in SiHa spheroids., Conclusions: Pre-clinical evaluation of PLGA-modified NP distribution into hypo-vascularized tumor tissue may benefit from considering tissue morphology in addition to cancer type.

More diverse multipurpose prevention technologies are urgently needed to provide localized, topical pre-exposure prophylaxis against sexually transmitted infections (STIs). In this work, we established the foundation for a multipurpose platform, in the form of polymeric electrospun fibers (EFs), to physicochemically treat herpes simplex virus 2 (HSV-2) infection. To initiate this study, we fabricated different formulations of poly(lactic-co-glycolic acid) (PLGA) and poly(dl-lactide-co-ε-caprolactone) (PLCL) EFs that encapsulate Acyclovir (ACV), to treat HSV-2 infection in vitro. Our goals were to assess the release and efficacy differences provided by these two different biodegradable polymers, and to determine how differing concentrations of ACV affected fiber efficacy against HSV-2 infection and the safety of each platform in vitro. Each formulation of PLGA and PLCL EFs exhibited high encapsulation efficiency of ACV, sustained-delivery of ACV through one month, and in vitro biocompatibility at the highest doses of EFs tested. Additionally, all EF formulations provided complete and efficacious protection against HSV-2 infection in vitro, regardless of the timeframe of collected fiber eluates tested. This work demonstrates the potential for PLGA and PLCL EFs as delivery platforms against HSV-2, and indicates that these delivery vehicles may be expanded upon to provide protection against other sexually transmitted infections., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite current prophylactic strategies, sexually transmitted infections (STIs) remain significant contributors to global health challenges, spurring the development of new multipurpose delivery technologies to protect individuals from and treat virus infections. However, there are few methods currently available to prevent and no method to date that cures human immunodeficiency virus (HIV) infection or combinations of STIs. While current oral and topical preexposure prophylaxes have protected against HIV infection, they have primarily relied on antiretrovirals (ARVs) to inhibit infection. Yet continued challenges with ARVs include user adherence to daily treatment regimens and the potential toxicity and antiviral resistance associated with chronic use. The integration of new biological agents may avert some of these adverse effects while also providing new mechanisms to prevent infection. Of the biologic-based antivirals, griffithsin (GRFT) has demonstrated potent inhibition of HIV-1 (and a multitude of other viruses) by adhering to and inactivating HIV-1 immediately upon contact. In parallel with the development of GRFT, electrospun fibers (EFs) have emerged as a promising platform for the delivery of agents active against HIV infection. In the study described here, our goal was to extend the mechanistic diversity of active agents and electrospun fibers by incorporating the biologic GRFT on the EF surface rather than within the EFs to inactivate HIV prior to cellular entry. We fabricated and characterized GRFT-modified EFs (GRFT-EFs) with different surface modification densities of GRFT and demonstrated their safety and efficacy against HIV-1 infection in vitro We believe that EFs are a unique platform that may be enhanced by incorporation of additional antiviral agents to prevent STIs via multiple mechanisms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Although a number of drugs have been developed for the treatment and prevention of human immunodeficiency virus (HIV) infection, it has proven difficult to optimize the drug and dosage parameters. The vaginal tissue, comprised of epithelial, stromal and blood compartments presents a complex system which challenges evaluation of drug kinetics solely through empirical effort. To provide insight into the underlying processes, mathematical modeling and computational simulation have been applied to the study of retroviral microbicide pharmacokinetics. Building upon previous pioneering work that modeled the delivery of Tenofovir (TFV) via topical delivery to the vaginal environment, here we computationally evaluate the performance of the retroviral inhibitor dapivirine released from a microbicide gel. We adapt the TFV model to simulate the multicompartmental diffusion and uptake of dapivirine into the blood plasma and vaginal compartments. The results show that dapivirine is expected to accumulate at the interface between the gel and epithelium compartments due to its hydrophobic characteristics. Hydrophobicity also results in decreased diffusivity, which may impact distribution by up to 2 orders of magnitude compared to TFV. Maximum concentrations of dapivirine in the epithelium, stroma, and blood were 9.9e7, 2.45e6, and 119pg/mL, respectively. This suggests that greater initial doses or longer time frames are required to obtain higher drug concentrations in the epithelium. These observations may have important ramifications if a specific time frame is required for efficacy, or if a minimum/maximum concentration is needed in the mucus, epithelium, or stroma based on combined efficacy and safety data., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Uncoordinated cellular proliferation and dysregulated angiogenesis in solid tumors are coupled with inadequate tissue, blood, and lymphatic vascularization. Consequently, tumors are often characterized by hypoxic regions with limited access to vascular-borne substances. In particular, systemically administered nanoparticles (NPs) targeting tumor cells and relying on vascular access to reach tumor tissue can suffer from limited therapeutic efficacy due to inhomogeneous intra-tumor distribution and insufficient cellular internalization of NPs. To circumvent these challenges, NP surfaces can be modified to facilitate tumor interstitial transport and cellular uptake., Results: We create poly(lactic-co-glycolic) acid NPs modified with MPG, polyethylene glycol (PEG), MPG/PEG, and Vimentin (VIM), and evaluate their cellular uptake in 2D (monolayer) cell culture of human cervical carcinoma (HeLa). We compare NP performance by evaluating uptake by non-cancerous vaginal (VK2) cells. We further assess NP interstitial transport in hypo-vascularized lesions by evaluating the effect of the various modifications on NP penetration in 3D cell culture of the HeLa cells. Results show that after 24 h incubation with HeLa cells in monolayer, MPG, MPG/PEG, PEG, and VIM NPs were internalized at 66×, 24×, 30×, and 15× that of unmodified NPs, respectively. In contrast, incubation with VK2 cells in monolayer showed that MPG , MPG/PEG , PEG , and VIM NPs internalized at 6.3×, 4.3×, 12.4×, and 3.0× that of unmodified NPs, respectively. Uptake was significantly enhanced in tumorigenic vs. normal cells, with internalization of MPG NPs by HeLa cells being twice that of PEG NPs by VK2 cells. After 24 h incubation in HeLa 3D cell culture, MPG and MPG/PEGNPs were internalized 2× and 3× compared to PEG and VIM NPs, respectively. Whereas MPG NPs were internalized mostly in the cell culture periphery (1.2×, 1.4×, and 2.7× that of PEG, MPG/PEG, and VIM NPs, respectively), PEG NPs at 250 μm penetrated 2× farther into the tissue culture than MPG NPs. For all NP types, cellular internalization was severely hindered in 3D compared to monolayer., Conclusions: Although MPG surface modification enhances internalization and uptake in hypo-vascularized cervical tissue culture, coating with PEG reduces this internalization while enhancing penetration. A delivery strategy combining NPs with either modification may balance cellular internalization vs. tissue penetration in hypo-vascularized cervical cancer lesions.