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.

Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

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.

Core-Cross-Linked Nanoparticles Reduce Neuroinflammation and Improve Outcome in a Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults, yet there are currently no treatments available that prevent the secondary spread of damage beyond the initial insult. The chronic progression of this secondary injury is in part caused by the release of reactive oxygen species (ROS) into surrounding normal brain. Thus, treatments that can enter the brain and reduce the spread of ROS should improve outcome from TBI. Here a highly versatile, reproducible, and scalable method to synthesize core-cross-linked nanoparticles (NPs) from polysorbate 80 (PS80) using a combination of thiol-ene and thiol-Michael chemistry is described. The resultant NPs consist of a ROS-reactive thioether cross-linked core stabilized in aqueous solution by hydroxy-functional oligoethylene oxide segments. These NPs show narrow molecular weight distributions and have a high proportion of thioether units that reduce local levels of ROS. In a controlled cortical impact mouse model of TBI, the NPs are able to rapidly accumulate and be retained in damaged brain as visualized through fluorescence imaging, reduce neuroinflammation and the secondary spread of injury as determined through magnetic resonance imaging and histopathology, and improve functional outcome as determined through behavioral analyses. Our findings provide strong evidence that these NPs may, upon further development and testing, provide a useful strategy to help improve the outcome of patients following a TBI.

Surface-enhanced Raman spectral biomarkers correlate with Ankle Brachial Index and characterize leg muscle biochemical composition of patients with peripheral arterial disease.

Peripheral arterial disease (PAD) is characterized by atherosclerotic blockages of the arteries supplying the lower extremities, which cause a progressive accumulation of ischemic injury to the skeletal muscles of the lower limbs. This injury includes altered metabolic processes, damaged organelles, and compromised bioenergetics in the affected muscles. The objective of this study was to explore the association of Raman spectral signatures of muscle biochemistry with the severity of atherosclerosis in the legs as determined by the Ankle Brachial Index (ABI) and clinical presentation. We collected muscle biopsies from the gastrocnemius (calf muscle) of five patients with clinically diagnosed claudication, five patients with clinically diagnosed critical limb ischemia (CLI), and five control patients who did not have PAD. A partial least squares regression (PLSR) model was able to predict patient ABI with a correlation coefficient of 0.99 during training and a correlation coefficient of 0.85 using a full cross-validation. When using the first three PLS factor scores in combination with linear discriminant analysis, the discriminant model was able to correctly classify the control, claudicating, and CLI patients with 100% accuracy, using a full cross-validation procedure. Raman spectroscopy is capable of detecting and measuring unique biochemical signatures of skeletal muscle. These signatures can discriminate control muscles from PAD muscles and correlate with the ABI and clinical presentation of the PAD patient. Raman spectroscopy provides novel spectral biomarkers that may complement existing methods for diagnosis and monitoring treatment of PAD patients.