Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at −20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.

An efficient shotgun lipidomics strategy was established and optimized for fast phospholipid profiling of viscera from three fish species: Lateolabrax japonicas, Ctenopharyngodon idellus, and Carassius auratus. This strategy relies on direct infusion of total lipid extracts into a tandem mass spectrometer without additional separation of the individual molecular species. Four classes of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), were analyzed, and at least 81 molecular species of phospholipids were identified, including 34 species of PC, 24 species of PE, 12 species of PS, and 11 species of PI, in both positive- and negative-ion electrospray ionization mode. The results show that fish viscera, which are traditionally discarded as fisheries wastes, are nutritional in phospholipids with total contents of the four detected phospholipid classes ranging from 1.52 to 3.29 mg/g in the three tested fish species. Regardless of the tested fish species, PC and PE are the dominant phospholipid classes, followed by PI and PS. Furthermore, principal component analysis (PCA) was applied to normalize the relative amounts of the identified phospholipid species. The results demonstrate that PS 18:0/22:6, PI 18:0/20:4, and PI 18:0/20:5 were the main contributors of cumulative value and could be used as an indicator for fish species differentiation. This shotgun lipidomics method was >10 times faster than traditional methods, because no chromatographic separation was needed. The successful application of this strategy paves the way for full utilization of traditionally discarded fisheries wastes and provides an alternative means for fish species differentiation.

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