Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy against Aerosolized Intracellular Alveolar Infections.

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating Franciscella tularensis pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16-17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal F.t. novicida mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding Cmax/MIC and AUC0-24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as F. tularensis, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles.

Nanostructured glycopolymer augmented liposomes to elucidate carbohydrate-mediated targeting.

Carbohydrate receptors on alveolar macrophages are attractive targets for receptor-mediated delivery of nanostructured therapeutics. In this study, we employed reversible addition fragmentation chain transfer polymerization to synthesize neoglycopolymers, consisting of mannose- and galactose methacrylate-based monomers copolymerized with cholesterol methacrylate for use in functional liposome studies. Glycopolymer-functional liposomes were employed to elucidate macrophage mannose receptor (CD206) and macrophage galactose-type lectin (CD301) targeting in both primary macrophage and immortal macrophage cell lines. Expression of CD206 and CD301 was observed to vary significantly between cell lines (murine alveolar macrophage, murine bone marrow-derived macrophage, RAW264.7, and MH-S), which has significant implications in in vitro targeting and uptake studies. Synthetic glycopolymers and glycopolymer augmented liposomes demonstrated specific receptor-mediated uptake in a manner dependent on carbohydrate receptor expression. These results establish a platform capable of probing endogenous carbohydrate receptor-mediated targeting via glycofunctional nanomaterials.

ASPIRE: A multi-site community-based participatory research project to increase understanding of the dynamics of violence against immigrant and refugee women in Australia.

BACKGROUND: One in three women around the world are or have been subjected to violence. This includes in Australia, where violence against women is an urgent public health and human rights issue. Immigrant and refugee women who have resettled in Australia are known to face barriers accessing services aimed at preventing and responding to family violence. However there is little evidence about the contexts, nature and dynamics of violence against immigrant and refugee women to inform appropriate responses to enhance their safety and well-being. The ASPIRE project will address this gap by identifying opportunities for the development of responsive local and community-based interventions for family violence against immigrant and refugee women, contributing to the currently limited Australian research in this area. METHODS/DESIGN: This participatory research project will work with communities in eight geographic locations (two inner-city, three outer-suburban, and three regional) across two states (Victoria and Tasmania), to generate evidence about immigrant and refugee women's experiences in a range of settings. The project will engage stakeholders and communities through extensive consultation prior to data collection and by facilitating community members' participation in generating and analysing data. A mix of qualitative methods will be used to generate rich data about the family, cultural and place-based contexts that shape the prevalence and dynamics of violence against immigrant and refugee women; women's prevention and help-seeking efforts; and community attitudes about and responses to violence across a range of cultural groups. Methods include in-depth interviews with women who have experienced family violence, key informant interviews with local community service providers, focus group discussions with men and women from predominant cultural groups that have migrated to areas covered by the research sites, and Photovoice with community leaders. Bilingual health educators will contribute to development of the research approach, the collection and analysis of data, and the dissemination of findings. DISCUSSION: Findings from this two-year study will be disseminated to communities, service providers and policy-makers, providing evidence to inform culturally-appropriate prevention and support interventions, and building local communities' awareness and capacity to respond to violence against immigrant and refugee women.

How structural and symbolic violence during resettlement impacts the social and mental wellbeing of forced migrant women: the lived experiences of Arabic speaking survivors of IPV resettled in Melbourne, Australia.

Forced migrant women experience high levels of violence across their journeys and violence can be characterised as having three overarching forms: structural, symbolic, and interpersonal. It is important to understand the intersecting nature of gendered forms of symbolic, structural and interpersonal violence, and their impact on the mental health of forced migrant women in order to develop holistic IPV and resettlement programs and interventions. This article adopts an ecological framework of violence and qualitative methods with mental health service providers and survivors of IPV to understand the intersections of different forms of violence and their impact on mental health as they relate to the lived experiences of Arabic-speaking forced migrant survivors currently residing in Melbourne, Australia. Our research has three key findings: (1) that forced migrant women living in Melbourne, Australia experience intersecting forms of violence during resettlement (2) Structural and symbolic violence against forced migrant women persists regardless of marital status (3) Autonomy and independence plays a vital role in the mental health and wellbeing of forced migrant women. Our findings reveal that structural and symbolic violence increase the risk of IPV for forced migrant women during resettlement and that even when forced migrant women leave IPV situations, structural and symbolic violence persist and exacerbate mental ill-health. This article also reveals the importance autonomy and independence in both the perpetration of violence and in healing and recovery.

Reproductive coercion as a form of family violence against immigrant and refugee women in Australia.

Reproductive coercion (RC), generally considered a form of intimate partner violence (IPV), refers to perpetrator behaviours and actions that are intended to interfere with and control the autonomous decision-making of a person regarding their reproductive health. To date there are few studies that document RC as experienced by immigrant and refugee women. In this article, we explore cases of RC as described by women who were part of a larger qualitative study investigating violence against immigrant and refugee women in southern Australia. The study aimed to identify the types of RC detailed in immigrant and refugee women's narratives, and to illustrate the contexts in which these experiences occurred. Analysis followed Baxter and Jack's (2008) case study methodology; whereby particular "cases" are used to describe a phenomenon in context. Thirteen women from seven countries described experiences that fit definitions of RC. The cases describe various types of RC including violence during pregnancy with the intent of causing miscarriage, forced abortion, contraception sabotage and forced pregnancy. As well as intimate partners, some women described multiple perpetrators being complicit in their experience of RC, especially in regard to controlling women's access to, and interactions with health services. More information is needed about immigrant and refugee women's experiences of RC, and how vulnerability to multi-perpetrator violence affects health service access. In particular knowledge about how multi-perpetrator RC can affect consent processes for women who already face barriers to health care requires attention. Further research is required to address knowledge gaps about appropriate prevention and advocacy work about RC in refugee and migrant communities, and what training is needed for professionals in the family violence sector, women's health services, women's organisations, multicultural and ethno-specific services.

Mannose Conjugated Polymer Targeting P. aeruginosa Biofilms.

Biofilms are one of the most challenging obstacles in bacterial infections. By providing protection against immune responses and antibiotic therapies, biofilms enable chronic colonization and the development of antibiotic resistance. As previous clinical observations and studies have shown, traditional antibiotic therapy alone cannot effectively treat and eliminate biofilm forming infections due to the protection conferred by the biofilm. A new strategy specifically targeting biofilms must be developed. Here, we specifically target and bind to the PAO1 biofilm and elucidate the molecular mechanism behind the interaction between a glycan targeted polymer and biofilm using a continuous flow biofilm model. The incubation of biofilms with fluorescent glycan targeted polymers demonstrated strong and persistent interactions with the mannose-containing polymer even after 24 h of continuous flow. To evaluate the role of major biofilm proteins LecB and CdrA, loss of function experiments with knockout variants established the dual involvement of both proteins in mannose targeted polymer retention. These results identify a persistent and specific targeting strategy to the biofilm, emphasizing its potential value as a delivery strategy and encouraging further exploration of biofilm targeted delivery.

Glycan targeted polymeric antibiotic prodrugs for alveolar macrophage infections.

Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis, can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages. We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection.

Polymer-augmented liposomes enhancing antibiotic delivery against intracellular infections.

Pulmonary intracellular infections, such as tuberculosis, anthrax, and tularemia, have remained a significant challenge to conventional antibiotic therapy. Ineffective antibiotic treatment of these infections can lead not only to undesired side effects, but also to the emergence of antibiotic resistance. Aminoglycosides (e.g., streptomycin) have long been part of the therapeutic regiment for many pulmonary intracellular infections. Their bioavailability for intracellular bacterial pools, however, is limited by poor membrane permeability and rapid elimination. To address this challenge, polymer-augmented liposomes (PALs) were developed to provide improved cytosolic delivery of streptomycin to alveolar macrophages, an important host cell for intracellular pathogens. A multifunctional diblock copolymer was engineered to functionalize PALs with carbohydrate-mediated targeting, pH-responsive drug release, and endosomal release activity with a single functional polymer that replaces the pegylated lipid component to simplify the liposome formulation. The pH-sensing functionality enabled PALs to provide enhanced release of streptomycin under endosomal pH conditions (70% release in 6 hours) with limited release at physiological pH 7.4 (16%). The membrane-destabilizing activity connected to endosomal release was characterized in a hemolysis assay and PALs displayed a sharp pH profile across the endosomal pH development target range. The direct connection of this membrane-destabilizing pH profile to model drug release was demonstrated in an established pyranine/p-xylene bispyridinium dibromide (DPX) fluorescence dequenching assay. PALs displayed similar sharp pH-responsive release, whereas PEGylated control liposomes did not, and similar profiles were then shown for streptomycin release. The mannose-targeting capability of the PALs was also demonstrated with 2.5 times higher internalization compared to non-targeted PEGylated liposomes. Finally, the streptomycin-loaded PALs were shown to have a significantly improved intracellular antibacterial activity in a Francisella-macrophage co-culture model, compared with free streptomycin or streptomycin delivered by control PEGylated liposomes (13× and 16×, respectively). This study suggests the potential of PALs as a useful platform to deliver antibiotics for the treatment of intracellular macrophage infections.

Macrophage-targeted drugamers with enzyme-cleavable linkers deliver high intracellular drug dosing and sustained drug pharmacokinetics against alveolar pulmonary infections.

Intracellular bacterial infections localized to the lung alveolar macrophage (AM) remain one of the most challenging settings for antimicrobial therapy. Current systemic antibiotic treatment fails to deliver sustained doses to intracellular bacterial reservoirs, which necessitates prolonged treatment regimens. Herein, we demonstrate a new intracellular enzyme-cleavable polymeric prodrug with tailored ciprofloxacin release profiles in the lungs and AM. The targeted polymeric prodrug, termed "drugamers", incorporates (1) hydrophilic mannose residues to solubilize the antibiotic cargo and to target and enhance AM uptake and intracellular delivery, and (2) enzyme-cleavable linkage chemistry to provide high and sustained intracellular AM drug dosing. Prodrug monomers, derived from the antibiotic ciprofloxacin, were synthesized with either an intracellular protease cleavable dipeptide linker or a hydrolytic phenyl ester linker. RAFT polymerization was used to copolymerize the prodrug monomers and mannose monomer to synthesize well-defined drugamers without requiring a post-polymerization conjugation step. In addition to favorable in vivo safety profiles following intratracheal administration, a single dose of the drugamers sustained ciprofloxacin dosing in lungs and AMs above the minimum inhibitory concentration (MIC) over at least a 48 h period. The enzyme-cleavable therapeutic achieved a >10-fold increase in sustained ciprofloxacin in AM, and maintained a significantly higher whole lung PK as well. Ciprofloxacin dosed in identical fashion displayed rapid clearance with a half-life of approximately 30 min. Notably, inhalation of the mannose-targeted ciprofloxacin drugamers achieved full survival (100%) in a highly lethal mouse model of pneumonic tularemia, contrasted with 0% survival using free ciprofloxacin. These findings demonstrate the versatility of the drugamer platform for engineering the intracellular pharmacokinetic profiles and its strong therapeutic activity in treating pulmonary intracellular infections.