Synthesis and Characterization of a Sustained Nitric Oxide-Releasing Orthodontic Elastomeric Chain for Antimicrobial Action.

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.

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.

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.

Computational Modeling of Antiviral Drug Diffusion from Poly(lactic- co-glycolic-acid) Fibers and Multicompartment Pharmacokinetics for Application to the Female Reproductive Tract.

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.

BAR-encapsulated nanoparticles for the inhibition and disruption of Porphyromonas gingivalis-Streptococcus gordonii biofilms.

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.

Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue.

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.

Griffithsin-Modified Electrospun Fibers as a Delivery Scaffold To Prevent HIV Infection.

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.

Enhanced uptake and transport of PLGA-modified nanoparticles in cervical cancer.

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.

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.

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.

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.

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.

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.

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.

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.