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Tuberculosis (TB) remains one of the most serious diseases caused by a single organism. Multiple (MDR) and extensively (XDR) drug resistant disease poses a threat to global health and requires new drugs and/or innovative approaches to treatment. A number of drugs have been proposed as inhaled therapy for TB, frequently prepared by spray drying. CPZEN-45 is a novel anti-tubercular drug that has poor oral bioavailability but has shown promise when administered via inhalation.Excipient-free CPZEN-45 HCl has been spray dried into a powder with physicochemical characteristics, aerodynamic particle size distribution, and delivered dose suitable for consideration as an inhaled product.The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the powder delivered using a RS01 inhaler were 2.62 ± 0.04 μm and 1.76 ± 0.09, respectively. Additionally, the powder was physically and chemically stable after storage at ambient conditions for1.5 years with particle size similar to freshly manufactured product. Overages in spray dried powder were recycled the powder and resprayed into drug product likewise resulting in negligible change in quality thus allowing for further preclinical characterization as necessary. CPZEN-45 was scaled up using pilot-scale manufacturing equipment where the density of the powder was increased to facilitate larger delivered doses without affecting the aerodynamic performance properties.The spray dried powders were suitable for pharmacokinetics, efficacy and preclinical toxicology studies. The final method of manufacture may be used directly for CGMP particle manufacture to support IND and Phase I clinical trials and beyond.

Phase-change contrast agents (PCCAs) consisting of lipid-encapsulated low-boiling point perfluorocarbons can be used in conjunction with ultrasound for diagnostic and therapeutic applications. One benefit of PCCAs is site-specific activation, whereby the liquid core is acoustically vaporized into a bubble detectable via ultrasound imaging. For further evaluation of PCCAs in a variety of applications, it is useful to disperse these nanodroplets into an acoustically compatible stationary matrix. However, many traditional phantom preparations require heating, which causes premature thermal activation of low-boiling point PCCAs. Polyvinyl alcohol (PVA) cryogels do not require heat to set. Here we propose a simple method for the incorporation of the low-boiling point PCCAs utilizing octafluoropropane (OFP) and decafluorbutane (DFB) into PVA cryogels for a variety of acoustic characterization applications. We then demonstrate the utility of the phantoms by activating droplets with a focused transducer, visualizing the lesions with ultrasound imaging. At 1 MHz, consistent droplet activation was consistently observed at 2.0 MPa and 4.0 MPa for OFP and DFB respectively.

Detection of bare gas microbubbles by magnetic resonance (MR) at low concentrations typically used in clinical contrast-ultrasound studies was recently demonstrated using hyperCEST. Despite the enhanced sensitivity achieved with hyperCEST, in vivo translation is challenging as on-resonance saturation of the gas-phase core of microbubbles consequently results in saturation of the gas-phase hyperpolarized

Tuberculosis (TB) remains the single most serious infectious disease attributable to a single-causative organism. A variety of drugs have been evaluated for pulmonary delivery as dry powders: capreomycin sulfate has shown efficacy and was safely delivered by inhalation at high doses to human volunteers, whereas CPZEN-45 is a new drug that has also been shown to kill resistant TB. The studies here combine these drugs—acting by different mechanisms—as components of single particles by spray-drying, yielding a new combination drug therapy. The spray-dried combination powder was prepared in an aerodynamic particle size range suitable for pulmonary delivery. Physicochemical storage stability was demonstrated for a period of 6 months. The spray-dried combination powders of capreomycin and CPZEN-45 have only moderate affinity for mucin, indicating that delivered drug will not be bound by these mucins in the lung and available for microbicidal effects. The pharmacokinetics of disposition in guinea pigs demonstrated high local concentrations of drug following direct administration to the lungs and subsequent systemic bioavailability. Further studies are required to demonstrate the in vivo efficacy of the combination to confirm the therapeutic potential of this novel combination.

IntroductionUltrasound is a relatively inexpensive and non-ionizing imaging modality, but is under-utilized in large airway assessments due to poor image quality. No commercially available contrast agents currently exist for sonographic evaluation of the respiratory system, nor has a respiratory route of microbubble contrast agent (MCA) administration been previously described for the enhancement of airway imaging.MethodsWe conducted a feasibility study to assess proof-of-concept for an inhalable ultrasound MCA composed of lipid-encapsulated decaflourobutane gas. The MCA was nebulized and administered as an aerosol through the lumen of an ex vivo porcine trachea, with image enhancement evaluated by comparing images pre- and post-exposure. Additionally, primary human bronchial epithelial (hBE) cells from three donors were differentiated at an air-liquid interface and exposed apically to 25 μL of undiluted MCA or vehicle control to assess contrast agent-induced cytotoxicity and inflammation. Basolateral medium was collected 24-hours post-exposure and lactate dehydrogenase (LDH) and interleukin-8 (IL-8) concentrations were measured as biomarkers of cytotoxicity and inflammation, respectively.ResultsContrast microbubbles remained intact following nebulization and enhanced sonographic delineation of ex vivo porcine tracheal walls, indicating adherence of the nebulized MCA to the lumenal mucosa. No significant cytotoxic or inflammatory effects were observed in cultured hBE cells following exposure to MCA.ConclusionsWe present proof-of-concept for an inhaled MCA for the enhancement of sonographic evaluations of the large airways. Pending further evaluations for safety and effectiveness, inhaled MCA may be feasible for clinical ultrasound applications, such as enhancing ultrasound-guided tracheal intubation, detecting airway bleeds, or monitoring large airway diseases in pediatric populations.

Gas microbubbles are an established clinical ultrasound contrast agent. They could also become a powerful MR intravascular contrast agent, but their low susceptibility-induced contrast requires high circulating concentrations or the addition of exogenous paramagnetic nanoparticles for MR detection. In order to detect clinical in vivo concentrations of raw microbubbles via MR, an alternative detection scheme must be used. HyperCEST is an NMR technique capable of indirectly detecting signals from very dilute molecules that exchange hyperpolarized (129)Xe and that are present at concentrations that fall well below the NMR detection threshold. Here, we use quantitative hyperCEST to show that microbubbles are very efficient hyperCEST agents. They can accommodate and saturate millions of (129)Xe atoms at a time, allowing for their indirect detection at concentrations as low as 10 femtomolar. The increased MR sensitivity to microbubbles achieved via hyperCEST can bridge the gap for microbubbles to become a dual modality contrast agent.

Pancreatic adenocarcinoma is an aggressive malignancy with limited therapeutic treatments available and a 5-y survival less than 10%. Pancreatic cancers have been found to be immunogenically "cold" with a largely immunosuppressive tumor microenvironment. There is emerging evidence that focused ultrasound can induce changes in the tumor microenvironment and have a constructive impact on the effect of immunotherapy. However, the immune cells and timing involved in these effects remain unclear, which is essential to determining how to combine immunotherapy with ultrasound for treatment of pancreatic adenocarcinoma. We used low-intensity focused ultrasound and microbubbles (LoFU + MBs), which can mechanically disrupt cellular membranes and vascular endothelia, to treat subcutaneous pancreatic tumors in C57BL/6 mice. To evaluate the immune cell landscape and expression and/or localization of damage-associated molecular patterns (DAMPs) as a response to ultrasound, we performed flow cytometry and histology on tumors and draining lymph nodes 2 and 15 d post-treatment. We repeated this study on larger tumors and with multiple treatments to determine whether similar or greater effects could be achieved. Two days after treatment, draining lymph nodes exhibited a significant increase in activated antigen presenting cells, such as macrophages, as well as expansion of CD8

Biofilms are associated with chronic infection and frequently require surgical intervention even after prolonged antibiotic therapy. Bactericidal antibiotics are often ineffective at eradicating genetically susceptible cells within a biofilm because: 1) most conventional antibiotics target ATP-dependent processes and so work poorly on metabolically indolent persister cells within a biofilm; and 2) the biofilm matrix can act as a physical barrier to drug diffusion of certain classes of antibiotics. Antibiotic therapy that fails to completely eradicate the pathogen leads to chronic and relapsing infections, major financial healthcare burdens and significant mortality. Numerous approaches have been taken to improve biofilm killing but these often fail to achieve eradication. We address this problem with a novel two-pronged strategy with the aim of eradicating biofilm infection using 1) antibiotics which target persister cells as well as 2) improving drug penetration using ultrasound-stimulated phase-change contrast agents (US-PCCA). We previously demonstrated that rhamnolipids, biosurfactant molecules produced by Pseudomonas aeruginosa, significantly potentiated aminoglycoside efficacy against S. aureus biofilm. We have also shown that US-PCCA can transiently disrupt biological barriers to therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could eradicate methicillin resistant S. aureus (MRSA) biofilms. To investigate this, we treated MRSA biofilms with a range of conventional and anti-persister antibiotics and/or US-PCCA. We found that vancomycin was significantly potentiated by US-PCCA, but a fraction of cells remained viable after the combination treatment. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically reduced biofilm viability, frequently culminating in complete eradication of the biofilm. These data demonstrate that biofilm eradication can be achieved using a combined approach improving drug penetration and therapeutics that target persister cells.

Routine exposure to inorganic nanoparticles (NPs) that are incorporated into consumer products such as foods/drinks, packaging materials, pharmaceuticals, and personal care products (e.g. cosmetics, sunscreens, shampoos) occurs on a daily basis. The standard everyday use of these products facilitates interactions between the incorporated inorganic NPs, mammalian tissues (e.g. skin, gastrointestinal tract, oral cavity), and the community of microbes that resides on these tissues. Changes to the microbiome have been linked to the initiation/ progression of many diseases and there is a growing interest focused on understanding how inorganic NPs can initiate these changes. As these mechanisms are revealed and defined, it may be possible to rationally design microbiotamodulating therapies based on inorganic NPs. In this article, we will: (i) provide a background on inorganic NPs that are commonly found in consumer products such as those that incorporate titanium, zinc, silver, silica, or iron, (ii) discuss how NP properties, microbiota composition, and the physiological microenvironment can mediate the effects that inorganic NPs have on the microbiota, and (iii) highlight opportunities for inorganic NP therapies that are designed to interact with, and navigate, the microbiome.

Phase change contrast agents (PCCAs) have been studied in the medical ultrasound field for nearly three decades. Their ability to convert from a liquid core droplet to an acoustically active microbubble has enhanced the possibilities of medical ultrasound, enabling new imaging approaches as well as therapeutic directions. However, traditional PCCAs are formulated with perfluorocarbons which are a liquid at standard temperature and pressure, requiring a high amount of energy to transition the encapsulated droplets to gas form, possibly resulting in undesired bioeffects. A new generation of low-boiling point PCCAs, which are formulated from gaseous perfluorocarbons in a metastable liquid state, seeks to overcome these limits. These super-heated liquid perfluorocarbon nanodroplets display longer circulation kinetics than microbubbles, their activation produces unique acoustic signatures, and their small particle size holds potential for extravascular applications. Low-boiling point nanodroplets can be phase-transitioned when activated with ultrasound at pressures and frequencies approved for diagnostic imaging. From the first publication almost 10 years ago, low-boiling point PCCA research has expanded rapidly, and recent advances in super-resolution imaging, drug delivery and neuromodulation made possible by these nanodroplets are just a few examples of this growing field of research. In this review, we discuss low-boiling point phase change contrast agents and their applications in ultrasound imaging and therapeutics.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration. We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.

Cavitation is an important phenomenon in biomedical acoustics. It can produce both desired outcomes (i.e., local therapeutic effects in vivo) and undesired outcomes (i.e., tissue damage), and it is, thus, important to both understand and direct cavitation fields. Through the use of three-dimensional-printed acoustic lenses and cavitation-sensitive acoustic phantoms, we demonstrate the generation of arbitrary shape two-dimensional (2D) microbubble cavitation fields. In this study, we demonstrate shaping a 1 MHz acoustic beam as the character “7” on a target plane that contains a higher mechanical index than the cavitation threshold for encapsulated microbubbles in a gelatin phantom. The lens pattern is first designed by calculating the phase map of the desired field using an angular spectrum approach. After lens implementation, acoustic pulsing through the lens generated the target acoustic field in a phantom and produced a cavitation map following the intended 2D pattern. The cavitation pattern was similar (with the structural similarity of 0.476) to the acoustic pressure map of the excitation beam.

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.