Messenger RNA is a class of promising nucleic acid therapeutics to treat a variety of diseases, including genetic diseases. The development of a stable and efficacious mRNA pulmonary delivery system would enable high therapeutic concentrations locally in the lungs to improve efficacy and limit potential toxicities. In this study, we employed a Design of Experiments (DOE) strategy to screen a library of lipid nanoparticle compositions to identify formulations possessing high potency both before and after aerosolization. Lipid nanoparticles (LNPs) showed stable physicochemical properties for at least 14 days of storage at 4 °C, and most formulations exhibited high encapsulation efficiencies greater than 80%. Generally, upon nebulization, LNP formulations showed increased particle size and decreased encapsulation efficiencies. An increasing molar ratio of poly-(ethylene) glycol (PEG)-lipid significantly decreased size but also intracellular protein expression of mRNA. We identified four formulations possessing higher intracellular protein expression ability in vitro even after aerosolization which were then assessed in in vivo studies. It was found that luciferase protein was predominately expressed in the mouse lung for the four lead formulations before and after nebulization. This study demonstrated that LNPs hold promise to be applied for aerosolization-mediated pulmonary mRNA delivery.

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This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., "biopanning"). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2-4 h. Compared to other phage enumeration approaches, qPCR is more timeefficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g., immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNAencapsulated viruses.

Recently, therapeutic uses of bacteriophage (phage) are gaining increased attention, yet common liquid phage formulations require cold chain storage that limits their potential use. Phage therapy is considered as an alternative to antibiotics for bacterial infections and more significantly a promising solution for the ever-increasing prevalence of multi-drug resistance (MDR) pathogens. One of the most promising applications of this therapy is to treat pulmonary bacterial infections. To efficiently deliver therapeutic phage to the lungs, phage formulations that allow for nebulization or dry powder inhalation are under active development. Several conventional particle engineering technologies have been applied in the development of dry powder inhalers (DPI), including spray drying, spray freeze drying, and atmospheric spray freeze drying, but these processes have their own disadvantages that limit their use with bacteriophage formulations and delivery. In our work, we hypothesize that thin film freeze-drying (TFFD) can be used to produce brittle matrix powders containing phage that may be suitable for delivery by several routes of administration, including by nebulization after reconstitution and by intranasal or inhalation delivery of the resulting dry powder. Here we selected T7 bacteriophage as our model phage in a preliminary screening study and found that a binary excipient matrix of sucrose and leucine at ratios of 80:20 or 75:25 by weight, protected bacteriophage from the stresses encountered during the TFFD process. In addition, we confirm that incorporating a buffer system during the TFFD process significantly improved the survival of phage during the ultra-rapid freezing step of the TFFD process and subsequent sublimation step in the lyophilization process. This preservation of phage bioactivity was significantly better than that observed for formulations without a buffer system. The titer loss of phage in standard SM buffer (Tris/NaCl/MgSO4/gelatin) containing formulation was as low as 0.2 log plaque forming units (pfu), which indicates that phage functionality was preserved after the TFFD process. Moreover, the presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicates that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating pulmonary delivery through DPIs. From these findings, we show that TFFD is a particle engineering method that can successfully produce phage containing powders that possess the desired properties for bioactivity and inhalation therapy.

Preparation in Materials and Methods) in the apical chamber of a 12 mm diameter (12 well) 109 polyester Transwell chamber with 3 micrometer pore size (Corning, Corning, NY). Before 110 addition of phage and mucin to the apical chamber, the basolateral chamber (i.e. donor 111 compartment) was filled with phosphate buffered saline without calcium and magnesium (PBS, 112 Corning, Corning, NY), as recommended by manufacturer. After 1 hour, peptide-presenting 113