Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles

Abstract

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.

Synthesis of monodisperse spherical AgNPs by ultrasound-intensified Lee-Meisel method, and quick evaluation via machine learning

“…Recently, it has been demonstrated that strong local shear stress generated by turbulence can promote the yield of platelet to enable clinical scale ex vivo manufacturing, which cannot be achieved in laminar flow [24] . The physico-chemical properties of nanocomposites and nanomedicine are also influenced dramatically by the competition between fluid dynamics and self-assembly performance [25] , [26] , [27] . With the help of ultrasound irradiation, Ag + could be reduced to AgNPs (around 8 nm) without using of any reductant and surfactant [28] .…”

Nanoparticle-mediated tumor cell expression of mIL-12 via systemic gene delivery treats syngeneic models of murine lung cancers

“…For in vivo experiments, nanoparticles comprised of the plasmid and l-PEI (in vivo-jetPEI, Polyplus Transfection, Illkirch, France) were prepared using the FNC process on a confined impinged jet (CIJ) device 14 , 16 . The CIJ device and all the fittings were autoclaved on a dry cycle prior to use.…”

“…Controlled by a digital syringe pump, the solutions were then steadily injected into the CIJ device at a flow rate of 20 mL/min. The turbulent mixing in the CIJ device allowed a superfast mixing of the materials, thus enabling controlled nanoparticle assembly kinetics that resulted in a controlled nanoparticle size and composition 14 . The production is an operator-independent process 16 that generated large quantities of nanoparticles with the same properties across different batches of studies.…”

“…Careful formulation and consideration of the size of these non-viral nanoparticles can also potentially enable a degree of selectivity from the perspective of enhanced permeability and retention (EPR) effect of tumors 12 , 13 . Precise control of the size and composition of l-PEI/DNA nanoparticles is possible using novel formulation techniques, such as flash nanocomplexation (FNC) 14 – 16 . Another critical aspect for systemic delivery of a genetic payload is the content of unmethylated CpG sequence, which triggers acute inframammary response as an innate immune reaction 17 – 19 .…”

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation

“…Whilst BPEI contains various primary, secondary, and tertiary amines, LPEI displays only secondary amines [ 11 , 13 ]. PEI is known for its high cationic charge density [ 14 ] enabling efficient condensation of nucleic acids via electrostatic interactions [ 10 , 15 , 16 , 17 ]. Most importantly, the high availability of amine groups of PEI displays a large buffering potential at physiological pH [ 14 ].…”